Phenyl-sulfamates as aromatase inhibitors

ABSTRACT

There is provided a compound of Formula I  
                 
 
wherein X, Y and Z are each independently of each other an optional linker group; R 1  is a ring system; R 2  is selected from hydrocarbyl groups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO 2 ) and halogens; R 3  and R 4  are independently selected from H and hydrocarbyl, ring A and B are independently optionally further substituted.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication PCT/GB2005/002194 filed Jun. 3, 2005 and published as WO2005/118560 on Dec. 15, 2005, which claims priority to Great BritainPatent Application No. 0412492.1 filed Jun. 4, 2004.

Each of the above referenced applications, and each document cited inthis text (“application cited documents”) and each document cited orreferenced in each of the application cited documents, and anymanufacturer's specifications or instructions for any products mentionedin this text and in any document incorporated into this text, are herebyincorporated herein by reference; and, technology in each of thedocuments incorporated herein by reference can be used in the practiceof this invention.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. Patent law; e.g., they canmean “includes”, “included”, “including” and the like. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in U.S. Patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the invention, and they exclude ingredients orsteps of the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, nonobvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. Patent law; namely, that these terms are closed ended.

Field of Invention

The present invention relates to a compound.

In particular the present invention relates to a compound and to apharmaceutical composition comprising the compound. The presentinvention also relates to the use of the compound or composition intherapy applications.

BACKGROUND TO THE INVENTION

Evidence suggests that oestrogens are the major mitogens involved inpromoting the growth of tumours in endocrine-dependent tissues, such asthe breast and endometrium. Although plasma oestrogen concentrations aresimilar in women with or without breast cancer, breast tumour oestroneand oestradiol levels are significantly higher than in normal breasttissue or blood. In situ synthesis of oestrogen is thought to make animportant contribution to the high levels of oestrogens in tumours andtherefore inhibitors, in particular specific inhibitors, of oestrogenbiosynthesis are of potential value for the treatment ofendocrine-dependent tumours.

Over the past two decades, there has been considerable interest in thedevelopment of inhibitors of the aromatase pathway—which converts theandrogen precursor androstenedione to oestrone. However, there is nowevidence that the oestrone sulphatase (E1-STS) pathway, i.e. thehydrolysis of oestrone sulphate to oestrone (E1S to E1), and aromatase(i.e. conversion of androstenedione to oestrone) account for theproduction of oestrogens in breast tumours.

FIGS. 1 and 2 are schematic diagrams showing some of the enzymesinvolved in the in situ synthesis of oestrone from oestrone sulphate,oestradiol and androstenedione.

In FIG. 2, which schematically shows the origin of oestrogenic steroidsin postmenopausal women, “ER” denotes Oestrogen Receptor, “DHEA-S”denotes Dehydroepiandrosterone-Sulphate, “Adiol” denotes Androstenediol,“E1-STS” denotes Oestrone Sulphatase, “DHEA-STS” denotesDHEA-sulphatase, “Adiol-STS” denotes Adiol Sulphatase, and “17B-HSD”denotes Oestradiol 17B-hydroxysteroid dehydrogenase.

As can be seen, the main two enzymes that are involved in the peripheralsynthesis of oestrogens are the aromatase enzyme and the enzyme oestronesulphatase.

In short, the aromatase enzyme converts androstenedione, which issecreted in large amounts by the adrenal cortex, to oestrone. Recentreports have suggested that some flavones could inhibit aromataseactivity.

Much of the oestrone so formed, however, is converted to oestronesulphate (E1S) and there is now a considerable body of evidence showingthat E1S in plasma and tissue acts as a reservoir for the formation ofoestrone by the action of oestrone sulphatase.

In this regard, it is now believed that the oestrone sulphatase (E1-STS)pathway—i.e. the hydrolysis of oestrone sulphate to oestrone (E1S to E1)is a major source of oestrogen in breast tumours. This theory issupported by a modest reduction of plasma oestrogen concentration inpostmenopausal women with breast cancer treated by aromatase inhibitors,such as aminoglutethimide and 4-hydroxyandrostenedione and also by thefact that plasma E1S concentration in these aromatase inhibitor-treatedpatients remains relatively high. The long half-life of E1S in blood(10-12 h) compared with the unconjugated oestrogens (20 min) and highlevels of steroid sulphatase activity in liver and, normal and malignantbreast tissues, also lend support to this theory.

Thus, oestrogen formation in malignant breast and endometrial tissuesvia the sulphatase pathway makes a major contribution to the highconcentration of oestrogens which are present in these tumours. However,inhibition of both the aromatase and sulphatase pathways could offerconsiderable therapeutic benefit.

PCT/GB92/01587 teaches novel steroid sulphatase inhibitors andpharmaceutical compositions containing them for use in the treatment ofoestrone dependent tumours, especially breast cancer. These steroidsulphatase inhibitors are sulphamate esters, such as N,N-dimethyloestrone-3-sulphamate and, preferably, oestrone-3-sulphamate (otherwiseknown as “EMATE”). EMATE has the following structure:

It is known that EMATE is a potent E1-STS inhibitor as it displays morethan 99% inhibition of E1-STS activity in intact MCF-7 cells at 0.1 nM.EMATE also inhibits the E1-STS enzyme in a time—andconcentration-dependent manner, indicating that it acts as an activesite-directed inactivator. Although EMATE was originally designed forthe inhibition of E1-STS, it also inhibits dehydroepiandrosteronesulphatase (DHEA-STS), which is an enzyme that is believed to have apivotal role in regulating the biosynthesis of the oestrogenic steroidandrostenediol. Also, there is now evidence to suggest thatandrostenediol may be of even greater importance as a promoter of breasttumour growth. EMATE is also active in vivo as almost completeinhibition of rat liver E1-STS (99%) and DHEA-STS (99%) activitiesresulted when it is administered either orally or subcutaneously. Inaddition, EMATE has been shown to have a memory enhancing effect inrats. Studies in mice have suggested an association between DHEA-STSactivity and the regulation of part of the immune response. It isthought that this may also occur in humans. The bridging O-atom of thesulphamate moiety in EMATE is important for inhibitory activity. Thus,when the 3-O-atom is replaced by other heteroatoms as inoestrone-3-N-sulphamate and oestrone-3-S-sulphamate, these analogues areweaker non-time-dependent inactivators.

In addition to oestrone, the other major steroid with oestrogenicproperties which is produced by postmenopausal women is androstenediol(see FIG. 2).

Androstenediol, although an androgen, can bind to the oestrogen receptor(ER) and can stimulate the growth of ER positive breast cancer cells andthe growth of carcinogen-induced mammary tumours in the rat.Importantly, in postmenopausal women 90% of the androstenediol producedoriginates from the androgen dehydroepiandrosterone sulphate (DHEA-S)which is secreted in large amounts by the adrenal cortex. DHEA-S isconverted to DHEA by DHEA sulphatase, which may be the same as, ordifferent from, the enzyme, oestrone sulphatase, which is responsiblefor the hydrolysis of E1S.

During the last 10-15 years considerable research has also been carriedout to develop potent aromatase inhibitors, some of which are nowmarketed. However, in three recent reports of postmenopausal women withbreast cancer who received aromatase inhibitor therapy, plasma E1Sconcentrations remained between 400-1000 pg/ml.

In summation therefore in situ synthesis of oestrogen is thought to makean important contribution to the high levels of oestrogens in tumoursand therefore specific inhibitors of oestrogen biosynthesis are ofpotential value for the treatment of endocrine-dependent tumours.

Moreover, even though oestrogen formation in malignant breast andendometrial tissues via the sulphatase pathway makes a majorcontribution to the high concentration of oestrogens, there are stillother enzymatic pathways that contribute to in vivo synthesis ofoestrogen.

Our earlier application WO03/045925 teaches compounds which may act asinhibitors of both aromatase and sulphatase. Many of the compounds ofthe disclosure are found to be extremely potent inhibitors of both ofthese enzymes. However, there is a desire to provide alternativecompounds or improved compounds.

The present invention seeks to provide novel compounds suitable for theinhibition of steroid sulphatase activity and aromatase activity.

SUMMARY ASPECTS OF THE PRESENT INVENTION

The present invention is based on the surprising finding that certainpolycyclic compounds could be used as effective steroid sulphataseinhibitors and/or aromatase inhibitors and/or as agents that caninfluence cell cycling and/or as agents that can influence apoptosis.

DETAILED ASPECTS OF THE PRESENT INVENTION

According to one aspect of the present invention, there is provided acompound of Formula I

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

The term “hydrocarbyl group” as used herein means a group comprising atleast C and H and may optionally comprise one or more other suitablesubstituents. Examples of such substituents may include halo, alkoxy,nitro, an alkyl group, a cyclic group etc. In addition to thepossibility of the substituents being a cyclic group, a combination ofsubstituents may form a cyclic group. If the hydrocarbyl group comprisesmore than one C then those carbons need not necessarily be linked toeach other. For example, at least two of the carbons may be linked via asuitable element or group. Thus, the hydrocarbyl group may containhetero atoms. Suitable hetero atoms will be apparent to those skilled inthe art and include, for instance, sulphur, nitrogen and oxygen. Anon-limiting example of a hydrocarbyl group is an acyl group.

The term “oxyhydrocarbyl” group as used herein means a group comprisingat least C, H and O and may optionally comprise one or more othersuitable substituents. Examples of such substituents may include halo-,alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to thepossibility of the substituents being a cyclic group, a combination ofsubstituents may form a cyclic group. If the oxyhydrocarbyl groupcomprises more than one C then those carbons need not necessarily belinked to each other. For example, at least two of the carbons may belinked via a suitable element or group. Thus, the oxyhydrocarbyl groupmay contain hetero atoms. Suitable hetero atoms will be apparent tothose skilled in the art and include, for instance, sulphur andnitrogen.

According to one aspect of the present invention, there is provided amethod comprising (a) performing a steroid sulphatase (STS) assay and/oraromatase assay with one or more candidate compounds defined herein; (b)determining whether one or more of said candidate compounds is/arecapable of modulating STS activity and/or aromatase activity and/or cellcycling and/or cell growth and/or apoptosis; and (c) selecting one ormore of said candidate compounds that is/are capable of modulating STSactivity and/or aromatase activity and/or cell cycling and/or cellgrowth and/or apoptosis.

According to one aspect of the present invention, there is provided amethod comprising (a) performing a steroid sulphatase assay and/oraromatase assay with one or more candidate compounds as defined herein;(b) determining whether one or more of said candidate compounds is/arecapable of inhibiting STS and/or aromatase activity; and (c) selectingone or more of said candidate compounds that is/are capable ofinhibiting STS activity and/or aromatase activity and/or cell cyclingand/or cell growth and/or apoptosis.

In any one of the methods of the present invention, one or moreadditional steps may be present. For example, the method may alsoinclude the step of modifying the identified candidate compound (such asby chemical and/or enzymatic techniques) and the optional additionalstep of testing that modified compound for STS inhibition effects (whichmay be to see if the effect is greater or different) and/or aromataseinhibition effects (which may be to see if the effect is greater ordifferent). By way of further example, the method may also include thestep of determining the structure (such as by use of crystallographictechniques) of the identified candidate compound and then performingcomputer modelling studies—such as to further increase its STS and/oraromatase inhibitory action. Thus, the present invention alsoencompasses a computer having a dataset (such as the crystallographicco-ordinates) for said identified candidate compound. The presentinvention also encompasses that identified candidate compound whenpresented on a computer screen for the analysis thereof—such as enzymeand/or protein binding studies.

According to one aspect of the present invention, there is provided acompound identified by the method of the present invention.

According to one aspect of the present invention, there is provided acompound according to the present invention for use in medicine.

According to one aspect of the present invention, there is provided apharmaceutical composition comprising the compound according to thepresent invention optionally admixed with a pharmaceutically acceptablecarrier, diluent, excipient or adjuvant.

According to one aspect of the present invention, there is provided theuse of a compound according to the present invention in the manufactureof a medicament for use in the therapy of a condition or diseaseassociated with STS and/or aromatase and/or cell cycling and/orapoptosis and/or cell growth.

According to one aspect of the present invention, there is provided theuse of a compound according to the present invention in the manufactureof a medicament for use in the therapy of a condition or diseaseassociated with adverse STS levels and/or adverse aromatase levelsand/or cell cycling and/or apoptosis and/or cell growth.

According to one aspect of the present invention, there is provided theuse of a compound according to the present invention in the manufactureof a medicament for inhibiting STS activity and/or inhibiting aromataseactivity.

According to one aspect of the present invention, there is provided theuse of a compound according to the present invention in the manufactureof a medicament for inhibiting STS activity and inhibiting aromataseactivity.

The present invention also encompasses the novel compounds of thepresent invention (such as those presented herein), as well as processesfor making same (such as the processes presented herein) as well asnovel intermediates (such as those presented herein) for use in thoseprocesses.

The compounds of the present invention may comprise other substituents.These other substituents may, for example, further increase the activityof the compounds of the present invention and/or increase stability (exvivo and/or in vivo).

For ease of reference, these and further aspects of the presentinvention are now discussed under appropriate section headings. However,the teachings under each section are not necessarily limited to eachparticular section.

Some Advantages

One key advantage of the present invention is that the compounds of thepresent invention can act as STS inhibitors.

One key advantage of the present invention is that the compounds of thepresent invention can act as aromatase inhibitors.

One key advantage of the present invention is that the compounds of thepresent invention can act as STS inhibitors and aromatase inhibitors.

Another advantage of the compounds of the present invention is that theymay be potent in vivo.

Some of the compounds of the present invention may be non-oestrogeniccompounds. Here, the term “non-oestrogenic” means exhibiting no orsubstantially no oestrogenic activity. Here, by the term“non-oestrogenic” means exhibiting no or substantially no systemicoestrogenic activity, such as that determined by Protocol 4.

Another advantage is that some of the compounds may not be capable ofbeing metabolised to compounds which display or induce hormonalactivity.

Some of the compounds of the present invention are also advantageous inthat they may be orally active.

Some of the compounds of the present invention may useful for theprevention and/or treatment of cancer, such as breast cancer, as well as(or in the alternative) non-malignant conditions, such as the preventionand/or treatment of inflammatory conditions—such as conditionsassociated with any one or more of: autoimmunity, including for example,rheumatoid arthritis, type I and II diabetes, systemic lupuserythematosus, multiple sclerosis, myasthenia gravis, thyroiditis,vasculitis, ulcerative colitis and Crohn's disease, skin disorders e.g.acne, psoriasis and contact dermatitis; graft versus host disease;eczema; asthma and organ rejection following transplantation. Thecompounds of the present invention are useful particularly whenpharmaceuticals may need to be administered from an early age.

Thus, some of the compounds of the present invention are also believedto have therapeutic uses other than for the treatment ofendocrine-dependent cancers, such as the treatment of autoimmunediseases.

The compounds of the present invention may also be useful as an inducerof apoptosis.

The compounds of the present invention may also be useful as a cellgrowth inhibitors.

Preferable Aspects

Hydrocarbyl Group

A typical hydrocarbyl group is a hydrocarbon group. Here the term“hydrocarbon” means any one of an alkyl group, an alkenyl group, analkynyl group, which groups may be linear, branched or cyclic, or anaryl group. The term hydrocarbon also includes those groups but whereinthey have been optionally substituted. If the hydrocarbon is a branchedstructure having substituent(s) thereon, then the substitution may be oneither the hydrocarbon backbone or on the branch; alternatively thesubstitutions may be on the hydrocarbon backbone and on the branch.

The hydrocarbyl/hydrocarbon/alkyl may be straight chain or branchedand/or may be saturated or unsaturated.

In one preferred aspect the hydrocarbyl/hydrocarbon/alkyl may beselected from straight or branched hydrocarbon groups containing atleast one hetero atom in the group.

In one preferred aspect the hydrocarbyl/hydrocarbon/alkyl may be ahydrocarbyl group comprising at least two carbons or wherein the totalnumber of carbons and hetero atoms is at least two.

In one preferred aspect the hydrocarbyl/hydrocarbon/alkyl may beselected from hydrocarbyl groups containing at least one hetero atom inthe group. Preferably the hetero atom is selected from sulphur, nitrogenand oxygen.

In one preferred aspect the hydrocarbyl/hydrocarbon/alkyl may beselected from straight or branched hydrocarbon groups containing atleast one hetero atom in the group. Preferably the hetero atom isselected from sulphur, nitrogen and oxygen.

In one preferred aspect the hydrocarbyl/hydrocarbon/alkyl may beselected from straight or branched alkyl groups, preferably C₁₋₁₀ alkyl,more preferably C₁₋₅ alkyl, containing at least one hetero atom in thegroup. Preferably the hetero atom is selected from sulphur, nitrogen andoxygen.

In one preferred aspect the hydrocarbyl/hydrocarbon/alkyl may beselected from straight chain alkyl groups, preferably C₁₋₁₀ alkyl, morepreferably C₁₋₅ alkyl, containing at least one hetero atom in the group.Preferably the hetero atom is selected from sulphur, nitrogen andoxygen.

The hydrocarbyl/hydrocarbon/alkyl may be selected from

-   -   C₁-C₁₀ hydrocarbyl,    -   C₁-C₅ hydrocarbyl    -   C₁-C₃ hydrocarbyl.    -   hydrocarbon groups    -   C₁-C₁₀ hydrocarbon    -   C₁-C₅ hydrocarbon    -   C₁-C₃ hydrocarbon.    -   alkyl groups    -   C₁-C₁₀ alkyl    -   C₁-C₅ alkyl    -   C₁-C₃ alkyl.

The hydrocarbyl/hydrocarbon/alkyl may be straight chain or branchedand/or may be saturated or unsaturated.

The hydrocarbyl/hydrocarbon/alkyl may be straight or branchedhydrocarbon groups containing at least one hetero atom in the group.

Oxyhydrocarbyl Group

In one embodiment of the present invention, the oxyhydrocarbyl group isa oxyhydrocarbon group.

Here the term “oxyhydrocarbon” means any one of an alkoxy group, anoxyalkenyl group, an oxyalkynyl group, which groups may be linear,branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon alsoincludes those groups but wherein they have been optionally substituted.If the oxyhydrocarbon is a branched structure having substituent(s)thereon, then the substitution may be on either the hydrocarbon backboneor on the branch; alternatively the substitutions may be on thehydrocarbon backbone and on the branch.

Each of the above teachings in respect of hydrocarbyl groups equallyapplies to the analogous oxyhydrocarbyl groups, that is thecorresponding oxyhydrocarbyl group which comprises an oxygen in additionto the hydrocarbyl.

Typically, the oxyhydrocarbyl group is of the formula C₁₋₆O (such as aC₁₋₃O).

Compound

In one preferred aspect the compound is of Formula II

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula III

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula IIIa

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula IV

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula V

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula VI

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula VIa

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula VII

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula VIIa

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula VIII

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.

In one preferred aspect the compound is of Formula VIIIa

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted.X, Y & Z

One of the more of the optional linker groups X, Y and Z may be present.In one aspect none of X, Y and Z are present.

In one preferred aspect at least one of the optional linker groups ispresent.

In one preferred aspect at least two of the optional linker groups arepresent.

In one preferred aspect each of X, Y and Z are present.

X

As discussed herein linker X is an optional group. In one aspect X ispresent. In one aspect X is not present. It will be appreciated by oneskilled in the art that when X is not present rings A and B are linkedvia a bond. Thus when X is not present the present invention provides acompound of Formula Ia

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; and ring Aand B are independently optionally further substituted. The aspectwherein X is not present is applicable to each of the preferred aspectsdescribed herein for example the aspect shown in preferred Formulae IIto VIII.

Preferably X is selected from hydrocarbyl, oxyhydrocarbyl, COO, CO, S,O, SO, SO₂, NR, and SO₂NR, wherein R is selected from H and hydrocarbylgroups.

When X is a hydrocarbyl group or in the option that X may be ahydrocarbyl group, preferably the hydrocarbyl group is a straight ofbranched alkyl group.

When X is a hydrocarbyl group or in the option that X may be ahydrocarbyl group, preferably the hydrocarbyl group is a straight chainalkyl group.

When X is a oxyhydrocarbyl group or in the option that X may be aoxyhydrocarbyl group, preferably the oxyhydrocarbyl group is —O-alkyl-,wherein alkyl is a straight of branched alkyl group.

When X is a oxyhydrocarbyl group or in the option that X may be aoxyhydrocarbyl group, preferably the oxyhydrocarbyl group is —O-alkyl-,wherein alkyl is a straight chain alkyl group

In one preferred aspect X is selected from groups selected from (CH₂)n,CH═CH (preferably trans configuration), O(CH₂)n, (CH₂)nO, S(CH₂)n,(CH₂)nS, CO(CH₂)n, (CH₂)nCO, CONH(CH₂)n, (CH₂)nCONH, COO(CH₂)n,(CH₂)nCOO, SO(CH₂)n, (CH₂)nSO, SO₂(CH₂)n, (CH₂)nSO₂, SO₂NC₁₋₆alkyl(CH₂)n(such as SO₂NMe(CH₂)n), (CH₂)nSO₂NC₁₋₆alkyl (such as (CH₂)nSO₂NMe);SO₂NH(CH₂)n, and (CH₂)nSO₂NH; wherein n is independently an integer from0 to 6. Preferably n is independently an integer from 1 to 6, morepreferably from 1 to 3, such as 1, 2 or 3.

In one preferred aspect X is selected from groups selected from (CH₂)n,O(CH₂)n, (CH₂)nO, S(CH₂)n, (CH₂)nS, CO(CH₂)n, (CH₂)nCO, CONH(CH₂)n,(CH₂)nCONH, COO(CH₂)n, (CH₂)nCOO, SO(CH₂)n, (CH₂)nSO, SO₂(CH₂)n,(CH₂)nSO₂, SO₂NH(CH₂)n, and (CH₂)nSO₂NH; wherein n is independently aninteger from 0 to 6. Preferably n is independently an integer from 1 to6, more preferably from 1 to 3, such as 1, 2 or 3.

In one preferred aspect X is selected from groups selected from SO₂NH,SO₂NMe, CONH, OCH₂, SCH₂, and CH═CH (preferably trans configuration).

Y

In one preferred aspect Y is selected from hydrocarbyl, oxyhydrocarbyl,COO, CO, S, O, SO, SO₂, NR, and SO₂NR, wherein R is selected from H andhydrocarbyl groups.

In one preferred aspect Y is selected from hydrocarbyl, CO, and SO₂.

When Y is a hydrocarbyl group or in the option that Y may be ahydrocarbyl group, preferably the hydrocarbyl group is a straight ofbranched alkyl group.

When Y is a hydrocarbyl group or in the option that Y may be ahydrocarbyl group, preferably the hydrocarbyl group is a straight chainalkyl group.

In one preferred aspect Y is selected from groups selected from (CH₂)m,CO(CH₂)m, (CH₂)mCO, SO₂(CH₂)m and wherein m is independently an integerfrom 0 to 6. Preferably m is independently an integer from 1 to 6, morepreferably from 1 to 3, such as 1, 2 or 3.

In a highly preferred aspect Y is (CH₂)m, wherein m is an integer from 0to 6, preferably an integer from 1 to 6, more preferably from 1 to 3,such as 1, 2 or 3. In a highly preferred aspect Y is —CH₂—.

Z

In one preferred aspect Z is selected from hydrocarbyl, oxyhydrocarbyl,COO, CO, S, O, SO, SO₂, NR, and SO₂NR, wherein R is selected from H andhydrocarbyl groups.

In one preferred aspect Z is a hydrocarbyl group.

When Z is a hydrocarbyl group or in the option that Z may be ahydrocarbyl group, preferably the hydrocarbyl group is a cycloalkylgroup. Preferably the cycloalkyl group is a C 3 to 10 cycloalkyl,preferably a C 3 to 6 cycloalkyl, most preferably a C3 or C4 cycloalkyl.

When Z is a hydrocarbyl group or in the option that Z may be ahydrocarbyl group, preferably the hydrocarbyl group is a straight ofbranched alkyl group.

When Z is a hydrocarbyl group or in the option that Z may be ahydrocarbyl group, preferably the hydrocarbyl group is a branched alkylgroup.

In one preferred aspect Z is CpH₂p, wherein p is an integer from 1 to 6.Preferably p is independently an integer from 1 to 3, such as 1, 2 or 3.

In one preferred aspect Z is a branched alkyl group of the formula CpH₂pand comprising at least one —C(CH₃)₂— moiety, wherein p is an integerfrom 1 to 6, preferably from 1 to 3, such as 1, 2 or 3.

In one preferred aspect Z is a —C(CH₃)₂— group.

In one preferred aspect Z is selected from —C(CH₃)₂—, —C(O)O—,

The ring system of R1 need not be a cyclic structure. In this regard,the ring system may be a linear structure that may have the ability toconform to a ring like structure when in in vivo. However in preferredaspects R1 is a cyclic structure.

R1 may be a heterocyclic group (a heterocycle) or a non-heterocyclicgroup. Suitable hetero atoms of a heterocyclic group include N, S and O.

When hetero atoms are present in a ring system to provide a heterocyclicgroup, the hetero atoms may be present in any amount. In one preferredaspect R1 is a ring system comprising carbon and one or more heteroatoms selected from N, S and O.

R1 may be is a saturated ring structure or an unsaturated ring structure(such as an aryl group).

Preferably, R1 is an aryl ring.

In one aspect of the invention R1 is selected from substituted orunsubstituted aromatic rings.

In one aspect R1 may be a polycyclic group, which need not be a fusedpolycycle. The term “polycyclic” includes fused and non-fused ringstructures including combinations thereof. If the ring system of R1 ispolycyclic some or all of the ring components of the ring system may befused together or joined via one or more suitable spacer groups.

The ring size of R1 may be chosen by one skilled in the art to achievecompounds having desired activity. Typically R1 is a ring systemcomprising from 3 to 10 members, such as ring systems comprising from 5,6 or 7 members.

Heterocyclic ring systems for use in the present invention includeimidazole, tetrazole, pyrazole, triazole, such as 1H-1,2,3-triazole,1H-1,2,4-triazole, 4H-1,2,4-triazole; optionally substituted 5- or6-membered heterocyclic group containing 1 to 3 hetero atoms eachselected from N, O and S, optionally substituted aryl (monocyclic orpolycyclic aromatic), pyridazine, pyrimidine, triazine such as 1,3,5triazine, and optionally substituted bicyclic condensed heterocyclicgroup consisting of the above heterocyclic group condensed with benzene.

In a highly preferred aspect R1 is selected from triazole, in particular1H-1,2,3-triazole, 1H-1,2,4-triazole, 4H-1,2,4-triazole

In a highly preferred aspect R1 is 1H-1,2,4-triazole.

In a highly preferred aspect R₁ is

R1 may be substituted by one or more substituents. Typical substituentsinclude hydrocarbyl, oxyhydrocarbyl, halo and cyano (—C≡N) groups. R1may also be substituted by one or more substituents selected fromphosphonate groups, thiophosphonate groups, sulphonate groups andsulphonamide groups.

In one preferred aspect R1 is unsubstituted.

—Y—R1

In a highly preferred Y is —CH₂— and R1 is

Thus in this aspect —Y—R1 together are the group

In one preferred aspect R₂ is selected from hydrocarbyl groups,oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens, whereinthe hydrocarbyl group is a straight of branched alkyl group.

When R2 is a hydrocarbyl group or in the option that R2 may be ahydrocarbyl group, preferably the hydrocarbyl group is a straight chainalkyl group.

When R2 is a hydrocarbyl group or in the option that R2 may be ahydrocarbyl group, preferably the hydrocarbyl group is (CH₂)qCH₃,wherein q is an integer from 0 to 6. Preferably q is an integer from 0to 3, such as 0, 1, 2 or 3.

In one preferred aspect R₂ is selected from hydrocarbyl groups,oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens, whereinthe oxyhydrocarbyl group is —O-alkyl-, wherein alkyl is a straight orbranched alkyl group.

When R2 is a oxyhydrocarbyl group or in the option that R2 may be aoxyhydrocarbyl group, preferably the oxyhydrocarbyl group is —O-alkyl-,wherein alkyl is a straight chain alkyl group

When R2 is a oxyhydrocarbyl group or in the option that R2 may be aoxyhydrocarbyl group, preferably the oxyhydrocarbyl group is—O(CH₂)rCH₃, wherein r is an integer from 0 to 6. Preferably r is aninteger from 0 to 3, such as 0, 1, 2 or 3.

In a highly preferred aspect R₂ is selected from —CH₃, —OCH₃, cyano(—CN), nitro (—NO₂) and halogens. The halogen may be selected from F,Cl, Br and I.

In a highly preferred aspect R₂ is selected from —CH₃, and —CN.

-Z-R2

In a highly preferred Z is selected from —C(CH₃)₂—, —C(O)O—,

or may be absent and R₂ is selected from —CH₃, and —CN.

Thus in this aspect -Z-R2 together may be selected from —C(CH₃)₂—CN,—C(O)O—CN, —CN,

R3 & R4

In one preferred aspect R₃ and R₄ are independently selected from H,alkyl, cycloalkyl, alkenyl, acyl and aryl, or combinations thereof, ortogether represent alkylene, wherein the or each alkyl or cycloalkyl oralkenyl or optionally contain one or more hetero atoms or groups.

In one preferred aspect at least one of R₃ and R₄ is H.

In one preferred aspect R₃ is H and R₄ is H.

Rings A & B

As noted herein the compounds of the present invention may compriseother substituents. These other substituents may, for example, furtherincrease the activity of the compounds of the present invention and/orincrease stability (ex vivo and/or in vivo). For example the ringdenoted A and B in the general formulae may comprise other substituents.However in one preferred aspect rings A and B are independently notfurther substituted.

In one aspect neither ring A nor ring B is further substituted.

In one aspect ring A is further substituted.

In one aspect ring B is further substituted.

In one preferred aspect ring A is further substituted and ring B is notfurther substituted.

If ring A and/or ring B is further substituted, the further substitutionmay be by groups selected from

-   -   hydrocarbyl groups, oxyhydrocarbyl groups, cyano (—CN), nitro        (—NO₂) and halogens.    -   C1-6 alkyl groups, C1-6 alkoxy groups, cyano (—CN), nitro (—NO₂)        and halogens.    -   —CH₃, —CH₂CH₃, —OCH₃, cyano (—CN), nitro (—NO₂) and halogens.

If ring A is further substituted, the further substitution is preferablya halogen and in particular, Cl, Br and/or F.

If ring A is further substituted, preferably ring A is substitution byonly one further substituent, that is preferably a halogen and inparticular, Cl, Br and/or F.

If ring A is further substituted, preferably the further substituent isat a position on the ring ortho to the sulphamate group.

Thus in one preferred aspect of the present invention, there is provideda compound of Formula Ib

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl; wherein R5is selected from hydrocarbyl groups, oxyhydrocarbyl groups, cyano (—CN),nitro (—NO₂) and halogens, and ring A and B are not further substituted.

Preferably R5 is selected from C1-6 alkyl groups, C1-6 alkoxy groups,cyano (—CN), nitro (—NO₂) and halogens. More preferably R5 is selectedfrom —CH₃, —CH₂CH₃, —OCH₃, cyano (—CN), nitro (—NO₂) and halogens. Morepreferably R5 is selected from Cl, Br and F.

Thus in one preferred aspect of the present invention, there is provideda compound of Formula Ic

wherein R₅ is as described herein.Further Preferred Compounds

In one preferred aspect the compound of the present invention may beselected from compounds of one of the formulae below. In the formulaebelow each ring may be substituted or unsubstituted or may contain oneor more additional bonds in the ring.

In one preferred aspect the compound of the present invention may beselected from compounds of the formula

wherein X is selected from groups selected from (CH₂)n, CH═CH(preferably trans configuration), O(CH₂)n, (CH₂)nO, S(CH₂)n, (CH₂)nS,CO(CH₂)n, (CH₂)nCO, CONH(CH₂)n, (CH₂)nCONH, COO(CH₂)n, (CH₂)nCOO,SO(CH₂)n, (CH₂)nSO, SO₂(CH₂)n, (CH₂)nSO₂, SO₂NC₁₋₆alkyl(CH₂)n (such asSO₂NMe(CH₂)n), (CH₂)nSO₂NC₁₋₆alkyl (such as (CH₂)nSO₂NMe); SO₂NH(CH₂)n,and (CH₂)nSO₂NH;; wherein n is independently an integer from 0 to 6,

wherein Y is selected from groups selected from (CH₂)m, CO(CH₂)m,(CH₂)mCO, SO₂(CH₂)m and wherein m is independently an integer from 0 to6;

wherein Z is selected from CpH₂p, C(O)O and C3 to 6 cycloalkyl groups,wherein p is an integer from 1 to 6; and

wherein R₂ is selected from —CH₃, —OCH₃, cyano (—CN), nitro (—NO₂) andhalogens.

In one preferred aspect the compound of the present invention may beselected from compounds of the formula

wherein X is selected from groups selected from (CH₂)n, O(CH₂)n,(CH₂)nO, S(CH₂)n, (CH₂)nS, CO(CH₂)n, (CH₂)nCO, CONH(CH₂)n, (CH₂)nCONH,COO(CH₂)n, (CH₂)nCOO, SO(CH₂)n, (CH₂)nSO, SO₂(CH₂)n, (CH₂)nSO₂,SO₂NH(CH₂)n, and (CH₂)nSO₂NH; wherein n is independently an integer from0 to 6,

wherein Y is selected from groups selected from (CH₂)m, CO(CH₂)m,(CH₂)mCO, SO₂(CH₂)m and wherein m is independently an integer from 0 to6;

wherein Z is CpH₂p, wherein p is an integer from 1 to 6; and

wherein R₂ is selected from —CH₃, —OCH₃, cyano (—CN), nitro (—NO₂) andhalogens.

A preferred compound of the present invention is a compound selectedfrom compounds of the formulae

A preferred compound of the present invention is a compound of theformula

Other Aspects

For some applications, preferably the compounds have no, or a minimal,oestrogenic effect.

For some applications, preferably the compounds have an oestrogeniceffect.

For some applications, preferably the compounds have a reversibleaction.

For some applications, preferably the compounds have an irreversibleaction.

In one embodiment, the compounds of the present invention are useful forthe treatment of breast cancer.

The present invention also covers novel intermediates that are useful toprepare the compounds of the present invention and metabolites of thecompounds of the present invention. For example, the present inventioncovers novel alcohol precursors for the compounds. By way of furtherexample, the present invention covers bis protected precursors for thecompounds. Examples of each of these precursors are presented herein.The present invention also encompasses a process comprising each or bothof those precursors for the synthesis of the compounds of the presentinvention.

Steroid Sulphatase

Steroid sulphatase—which is sometimes referred to as steroid sulphataseor steryl sulphatase or “STS” for short—hydrolyses several sulphatedsteroids, such as oestrone sulphate, dehydroepiandrosterone sulphate andcholesterol sulphate. STS has been allocated the enzyme number EC3.1.6.2.

STS has been cloned and expressed. For example see Stein et al (J. Biol.Chem. 264:13865-13872 (1989)) and Yen et al (Cell 49:443-454(1987)).

STS is an enzyme that has been implicated in a number of diseaseconditions.

By way of example, workers have found that a total deficiency in STSproduces ichthyosis. According to some workers, STS deficiency is fairlyprevalent in Japan. The same workers (Sakura et al, J Inherit Metab Dis1997 November;20(6):807-10) have also reported that allergicdiseases—such as bronchial asthma, allergic rhinitis, or atopicdermatitis—may be associated with a steroid sulphatase deficiency.

In addition to disease states being brought on through a total lack ofSTS activity, an increased level of STS activity may also bring aboutdisease conditions. By way of example, and as indicated above, there isstrong evidence to support a role of STS in breast cancer growth andmetastasis.

STS has also been implicated in other disease conditions. By way ofexample, Le Roy et al (Behav Genet 1999 March;29(2):131-6) havedetermined that there may be a genetic correlation between steroidsulphatase concentration and initiation of attack behaviour in mice. Theauthors conclude that sulphatation of steroids may be the prime mover ofa complex network, including genes shown to be implicated in aggressionby mutagenesis.

STS Inhibition

It is believed that some disease conditions associated with STS activityare due to conversion of a nonactive, sulphated oestrone to an active,nonsulphated oestrone. In disease conditions associated with STSactivity, it would be desirable to inhibit STS activity.

Here, the term “inhibit” includes reduce and/or eliminate and/or maskand/or prevent the detrimental action of STS.

STS Inhibitor

In accordance with the present invention, the compound of the presentinvention is capable of acting as an STS inhibitor.

Here, the term “inhibitor” as used herein with respect to the compoundof the present invention means a compound that can inhibit STSactivity—such as reduce and/or eliminate and/or mask and/or prevent thedetrimental action of STS. The STS inhibitor may act as an antagonist.

The ability of compounds to inhibit oestrone sulphatase activity can beassessed using either intact JEG3 choriocarcinoma cells or placentalmicrosomes. In addition, an animal model may be used. Details onsuitable Assay Protocols are presented in following sections. It is tobe noted that other assays could be used to determine STS activity andthus STS inhibition. For example, reference may also be made to theteachings of WO-A-99/50453.

In one aspect, for some applications, the compound is furthercharacterised by the feature that if the sulphamate group were to besubstituted by a sulphate group to form a sulphate derivative, then thesulphate derivative would be hydrolysable by an enzyme having steroidsulphatase (E.C. 3.1.6.2) activity—i.e. when incubated with steroidsulphatase EC 3.1.6.2 at pH 7.4 and 37° C.

In one preferred embodiment, if the sulphamate group of the compoundwere to be replaced with a sulphate group to form a sulphate compoundthen that sulphate compound would be hydrolysable by an enzyme havingsteroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value ofless than 200 mmolar, preferably less than 150 mmolar, preferably lessthan 100 mmolar, preferably less than 75 mmolar, preferably less than 50mmolar, when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and37° C.

For some applications, preferably the compound of the present inventionhas at least about a 100 fold selectivity to a desired target (e.g. STSand/or aromatase), preferably at least about a 150 fold selectivity tothe desired target, preferably at least about a 200 fold selectivity tothe desired target, preferably at least about a 250 fold selectivity tothe desired target, preferably at least about a 300 fold selectivity tothe desired target, preferably at least about a 350 fold selectivity tothe desired target.

It is to be noted that the compound of the present invention may haveother beneficial properties in addition to or in the alternative to itsability to inhibit STS and/or aromatase activity.

Sulphamate Group

The term “sulphamate” as used herein includes an ester of sulphamicacid, or an ester of an N-substituted derivative of sulphamic acid, or asalt thereof.

If R³ is a sulphamate group then the compound of the present inventionis referred to as a sulphamate compound.

Typically, the sulphamate group has the formula:(R₃)(R₄)N—S(O)(O)—O—wherein preferably R₃ and R₄ are independently selected from H, alkyl,cycloalkyl, alkenyl, acyl and aryl, or combinations thereof, or togetherrepresent alkylene, wherein the or each alkyl or cycloalkyl or alkenylor optionally contain one or more hetero atoms or groups.

When substituted, the N-substituted compounds of this invention maycontain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-arylsubstituents, preferably containing or each containing a maximum of 10carbon atoms.

When R₃ and/or R₄ is hydrocarbyl, the preferred values are those whereR₃ and R₄ are each independently selected C₁-C₁₀ hydrocarbyl, C₁-C₅hydrocarbyl, C₁-C₃ hydrocarbyl, C₁-C₁₀ hydrocarbon, C₁-C₅ hydrocarbon,C₁-C₃ hydrocarbon, C₁-C₁₀ alkyl, C₁-C₅ alkyl and C₁-C₃ alkyl.

When R₃ and/or R₄ is alkyl, the preferred values are those where R₃ andR₄ are each independently selected from lower alkyl groups containingfrom 1 to 6 carbon atoms, that is to say methyl, ethyl, propyl etc. R₃and R₄ may both be methyl.

When R₃ and/or R₄ is aryl, typical values are phenyl and tolyl (PhCH₃;o).

Where R₃ and/or R₄ represent cycloalkyl, typical values are cyclopropyl,cyclopentyl, cyclohexyl etc.

When joined together R₃ and R₄ typically represent an alkylene groupproviding a chain of 4 to 6 carbon atoms, optionally interrupted by oneor more hetero atoms or groups, e.g. to provide a 5 memberedheterocycle, e.g. morpholino, pyrrolidino or piperidino.

Within the values alkyl, cycloalkyl, alkenyl, acyl and aryl substitutedgroups are included containing as substituents therein one or moregroups which do not interfere with the sulphatase inhibitory activity ofthe compound in question. Exemplary non-interfering substituents includehydroxy, amino, halo, alkoxy, alkyl and aryl.

In some embodiments, the sulphamate group may form a ring structure bybeing fused to (or associated with) one or more atoms in or on ring A.

In some embodiments, there may be more than one sulphamate group. By wayof example, there may be two sulphamates (i.e. bis-sulphamatecompounds).

In some preferred embodiments, at least one of R₃ and R₄ is H.

In some further preferred embodiments, each of R₃ and R₄ is H.

Other Substituents

The compound of the present invention may have substituents other thanthose of formula I. By way of example, these other substituents may beone or more of: one or more sulphamate group(s), one or more phosphonategroup(s), one or more thiophosphonate group(s), one or more sulphonategroup(s), one or more sulphonamide group(s), one or more halo groups,one or more O groups, one or more hydroxy groups, one or more aminogroups, one or more sulphur containing group(s), one or more hydrocarbylgroup(s)—such as an oxyhydrocarbyl group.

Assay for Determining STS Activity Using Cancer Cells Protocol 1

Inhibition of Steroid Sulphatase Activity in JEG3 Cells

Steroid sulphatase activity is measured in vitro using intact JEG3choriocarcinoma cells. This cell line may be used to study the controlof human breast cancer cell growth. It possesses significant steroidsulphatase activity (Boivin et al., J. Med. Chem., 2000, 43: 4465-4478)and is available in from the American Type Culture Collection (ATCC).

Cells are maintained in Minimal Essential Medium (MEM) (FlowLaboratories, Irvine, Scotland) containing 20 mM HEPES, 5% foetal bovineserum, 2 mM glutamine, non-essential amino acids and 0.075% sodiumbicarbonate. Up to 30 replicate 25 cm2 tissue culture flasks are seededwith approximately 1×10⁵ cells/flask using the above medium. Cells aregrown to 80% confluency and the medium is changed every third day.

Intact monolayers of JEG3 cells in triplicate 25 cm² tissue cultureflasks are washed with Earle's Balanced Salt Solution (EBSS from ICNFlow, High Wycombe, U.K.) and incubated for 3-4 hours at 37° C. with 5pmol (7×10⁵ dpm) [6,7-3H]oestrone-3-sulphate (specific activity 60Ci/mmol from New England Nuclear, Boston, Mass., U.S.A.) in serum-freeMEM (2.5 ml) together with oestrone-3-sulphamate (11 concentrations: 0;1 fM; 0.01 pM; 0.1 pM; 1 pM; 0.01 nM; 0.1 nM; 1 nM; 0.01 mM; 0.1 mM; 1mM). After incubation each flask is cooled and the medium (1 ml) ispipetted into separate tubes containing [14C]oestrone (7×103 dpm)(specific activity 97 Ci/mmol from Amersham International RadiochemicalCentre, Amersham, U.K.). The mixture is shaken thoroughly for 30 secondswith toluene (5 ml). Experiments have shown that >90% [14C] oestrone and<0.1% [3H]oestrone-3-sulphate is removed from the aqueous phase by thistreatment. A portion (2 ml) of the organic phase is removed, evaporatedand the 3H and 14C content of the residue determined by scintillationspectrometry. The mass of oestrone-3-sulphate hydrolysed was calculatedfrom the 3H counts obtained (corrected for the volumes of the medium andorganic phase used, and for recovery of [14C] oestrone added) and thespecific activity of the substrate. Each batch of experiments includesincubations of microsomes prepared from a sulphatase-positive humanplacenta (positive control) and flasks without cells (to assess apparentnon-enzymatic hydrolysis of the substrate). The number of cell nucleiper flask is determined using a Coulter Counter after treating the cellmonolayers with Zaponin. One flask in each batch is used to assess cellmembrane status and viability using the Trypan Blue exclusion method(Phillips, H. J. (1973) In: Tissue culture and applications, [eds:Kruse, D. F. & Patterson, M. K.]; pp. 406-408; Academic Press, NewYork).

Results for steroid sulphatase activity are expressed as the mean±1 S.D.of the total product (oestrone+oestradiol) formed during the incubationperiod (3-4 hours) calculated for 106 cells and, for values showingstatistical significance, as a percentage reduction (inhibition) overincubations containing no oestrone-3-sulphamate. Unpaired Student'st-test was used to test the statistical significance of results.

Assay for Determining STS Activity Using Placental Microsomes Protocol 2

Inhibition of Steroid Sulphatase Activity in Placental Microsomes

Sulphatase-positive human placenta from normal term pregnancies arethoroughly minced with scissors and washed once with cold phosphatebuffer (pH 7.4, 50 mM) then re-suspended in cold phosphate buffer (5ml/g tissue). Homogenisation is accomplished with an Ultra-Turraxhomogeniser, using three 10 second bursts separated by 2 minute coolingperiods in ice. Nuclei and cell debris are removed by centrifuging (4°C.) at 2000 g for 30 minutes and portions (2 ml) of the supernatant arestored at 20° C. The protein concentration of the supernatants isdetermined by the method of Bradford (Anal. Biochem., 72, 248-254(1976)).

Incubations (1 ml) are carried out using a protein concentration of 100mg/ml, substrate concentration of 20 mM [6,7-3H]oestrone-3-sulphate(specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass.,U.S.A.) and an incubation time of 20 minutes at 37° C. If necessaryeight concentrations of compounds are employed: 0 (i.e. control); 0.05mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1.0 mM. After incubationeach sample is cooled and the medium (1 ml) was pipetted into separatetubes containing [14C]oestrone (7×103 dpm) (specific activity 97 Ci/mmolfrom Amersham International Radiochemical Centre, Amersham, U.K.). Themixture is shaken thoroughly for 30 seconds with toluene (5 ml).Experiments have shown that >90% [14C]oestrone and <0.1%[3H]oestrone-3-sulphate is removed from the aqueous phase by thistreatment. A portion (2 ml) of the organic phase was removed, evaporatedand the 3H and 14C content of the residue determined by scintillationspectrometry. The mass of oestrone-3-sulphate hydrolysed is calculatedfrom the 3H counts obtained (corrected for the volumes of the medium andorganic phase used, and for recovery of [14C]oestrone added) and thespecific activity of the substrate.

Animal Assay Model for Determining STS Activity Protocol 3

Inhibition of Oestrone Sulphatase Activity in vivo

The compounds of the present invention may be studied using an animalmodel, in particular in ovariectomised rats. In this model compoundswhich are oestrogenic stimulate uterine growth.

The compound (0.1 mg/Kg/day for five days) is administered orally torats with another group of animals receiving vehicle only (propyleneglycol). At the end of the study samples of liver tissue were obtainedand oestrone sulphatase activity assayed using 3H oestrone sulphate asthe substrate as previously described (see PCT/GB95/02638).

Animal Assay Model for Determining Oestrogenic Activity Protocol 4

The compounds of the present invention may be studied using an animalmodel, in particular in ovariectomised rats. In this model, compoundswhich are oestrogenic stimulate uterine growth.

The compound (0.1 mg/Kg/day for five days) was administered orally torats with another group of animals receiving vehicle only (propyleneglycol). At the end of the study uteri were obtained and weighed withthe results being expressed as uterine weight/whole body weight×100.

Compounds having no significant effect on uterine growth are notoestrogenic.

Biotechnological Assays for Determining STS Activity Protocol 5

The ability of compounds to inhibit oestrone sulphatase activity canalso be assessed using amino acid sequences or nucleotide sequencesencoding STS, or active fragments, derivatives, homologues or variantsthereof in, for example, high-through put screens. Such assays andmethods for their pratice are taught in WO 03/045925 which isincorporated herein by reference.

In one preferred aspect, the present invention relates to a method ofidentifying agents that selectively modulate STS, which compounds havethe formula (I).

Assay for Determining Aromatase Activity Using JEG3 Cells Protocol 6

Aromatase activity is measured in JEG3 choriocarcinoma cells, obtainedfrom the ATCC. This cell line possesses significant aromatase activityand is widely used to study the control of human aromatase activity(Bhatnager et al., J. Steroid Biochem. Molec. Biol. 2001, 76: 199-202).Cells are maintained in Minimal Essential Medium (MEM, FlowLaboratories, Irvine, Scotland) containing 20 mM HEPES, 10% foetalbovine serum, 2 mM glutamine, non-essential amino acids and 0.075%sodium bicarbonate. Intact monolayers of JEG3 cells (2.5×10⁶ cells) intriplicate 25 cm² tissue culture flasks are washed with Earle's Balancedsalt solution (EBSS, from ICN Flow, High Wycombe, UK) and incubated with[1β-³H] androstenedione (2-5 nM, 26 Ci/mmol, New England Nuclear,Boston, Mass., USA) for 30 min with inhibitors over the range of 10pm-10 μM. During the aromatase reaction, ³H₂O is liberated which can hequantified using a liquid scintillation spectrometer (Beckman-Coulter,High Wycombe, Bucks. UK). This ³H₂O-release method has been widely usedto measure aromatase activity (Newton et al., J.Steroid Biochem.1986,24: 1033-1039). The number of cell nuclei per flask is determinedusing a Coulter Counter after treating the cell monolayers with Zaponin.

Results for aromatase activity are expressed as the mean ±1 S.D. of theproduct formed during the incubation period (30 min) calculated for 10⁶cells and, for values showing a statistical significance, as apercentage reduction (inhibition) over incubations containing noaromatase inhibitor. Unpaired Student's t test was used to test thestatistical significance of results. IC₅₀ values were calculated as theconcentration of inhibitor required to obtain a 50% inhibition ofaromatase activity.

Animal Assays for Determining Aromatase Activity Protocol 7

(i) Inhibition of PMSG-induced Oestrogen Synthesis

The ability of compounds to inhibit aromatase activity in vivo wastested using a pregnant mare serum gonadotrophin (PMSG)-inducedoestrogen synthesis assay. For this, female rats (250 g) were injectedwith PMSG (200 IU, s.c.). After 72 h rats were administered vehicle(propylene glycol) or various doses of test compounds orally. At 2 hafter dosing blood samples were obtained by cardiac puncture (underanaesthesia). Plasma oestradiol levels were measured in control groupsand groups receiving drugs. The efficacy of aromatase inhibition wasdetermined by measurement of plasma oestradiol concentrations byradioimmunoassay. This method has been widely used to determine theeffectiveness of aromatase inhibitors in vivo (Wouters et al., J.SteroidBiochem., 1989, 32: 781-788).

(ii) Inhibition of Androstenedione Stimulated Uterine Growth inOvariectomised Rats

Female rats (250 g) were ovariectomised and used to determine theeffectiveness of aromatase inhibition on androstenedione stimulateduterine growth. Administration of androstenedione (30 mg/kg/d) for a2-week period results in a significant increase in uterine growth inovariectomised animals. This increase in uterine growth is stimulated byoestrogen which is derived from the administered androstenedione as aresult of the action of the aromatase enzyme. By co-administration ofcompounds with androstenedione the extent of aromatase inhibition can bedetermined by measurements of uterine weights in treated and untreatedanimals.

Therapy

The compounds of the present invention may be used as therapeuticagents—i.e. in therapy applications.

The term “therapy” includes curative effects, alleviation effects, andprophylactic effects.

The therapy may be on humans or animals, preferably female animals.

Pharmaceutical Compositions

In one aspect, the present invention provides a pharmaceuticalcomposition, which comprises a compound according to the presentinvention and optionally a pharmaceutically acceptable carrier, diluentor excipient (including combinations thereof).

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes.

Where the agent is to be delivered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit though the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

Combination Pharmaceutical

The compound of the present invention may be used in combination withone or more other active agents, such as one or more otherpharmaceutically active agents.

By way of example, the compounds of the present invention may be used incombination with other STS inhibitors and/or other inhibitors such as anaromatase inhibitor (such as for example, 4-hydroxyandrostenedione(4-OHA)) and/or steroids—such as the naturally occurring neurosteroidsdehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS)and/or other structurally similar organic compounds. Examples of otherSTS inhibitors may be found in the above references. By way of example,STS inhibitors for use in the present invention include EMATE, andeither or both of the 2-ethyl and 2-methoxy 17-deoxy compounds that areanalogous to compound 5 presented herein.

In addition, or in the alternative, the compound of the presentinvention may be used in combination with a biological responsemodifier.

The term biological response modifier (“BRM”) includes cytokines, immunemodulators, growth factors, haematopoiesis regulating factors, colonystimulating factors, chemotactic, haemolytic and thrombolytic factors,cell surface receptors, ligands, leukocyte adhesion molecules,monoclonal antibodies, preventative and therapeutic vaccines, hormones,extracellular matrix components, fibronectin, etc. For someapplications, preferably, the biological response modifier is acytokine. Examples of cytokines include: interleukins (IL)—such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-19; Tumour Necrosis Factor (TNF)—such as TNF-α; Interferon alpha,beta and gamma; TGF-β. For some applications, preferably the cytokine istumour necrosis factor (TNF). For some applications, the TNF may be anytype of TNF—such as TNF-α, TNF-β, including derivatives or mixturesthereof. More preferably the cytokine is TNF-α. Teachings on TNF may befound in the art—such as WO-A-98/08870 and WO-A-98/13348.

Administration

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject and it will vary with the age,weight and response of the particular patient. The dosages below areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited.

The compositions of the present invention may be administered by directinjection. The composition may be formulated for parenteral, mucosal,intramuscular, intravenous, subcutaneous, intraocular or transdermaladministration. Depending upon the need, the agent may be administeredat a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

By way of further example, the agents of the present invention may beadministered in accordance with a regimen of 1 to 4 times per day,preferably once or twice per day. The specific dose level and frequencyof dosage for any particular patient may be varied and will depend upona variety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the host undergoing therapy.

Aside from the typical modes of delivery—indicated above—the term“administered” also includes delivery by techniques such as lipidmediated transfection, liposomes, immunoliposomes, lipofectin, cationicfacial amphiphiles (CFAs) and combinations thereof. The routes for suchdelivery mechanisms include but are not limited to mucosal, nasal, oral,parenteral, gastrointestinal, topical, or sublingual routes.

The term “administered” includes but is not limited to delivery by amucosal route, for example, as a nasal spray or aerosol for inhalationor as an ingestable solution; a parenteral route where delivery is by aninjectable form, such as, for example, an intravenous, intramuscular orsubcutaneous route.

Thus, for pharmaceutical administration, the STS inhibitors of thepresent invention can be formulated in any suitable manner utilisingconventional pharmaceutical formulating techniques and pharmaceuticalcarriers, adjuvants, excipients, diluents etc. and usually forparenteral administration. Approximate effective dose rates may be inthe range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or evenfrom 100 to 800 mg/day depending on the individual activities of thecompounds in question and for a patient of average (70 Kg) bodyweight.More usual dosage rates for the preferred and more active compounds willbe in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day,most preferably from 200 to 250 mg/day. They may be given in single doseregimes, split dose regimes and/or in multiple dose regimes lasting overseveral days. For oral administration they may be formulated in tablets,capsules, solution or suspension containing from 100 to 500 mg ofcompound per unit dose. Alternatively and preferably the compounds willbe formulated for parenteral administration in a suitable parenterallyadministrable carrier and providing single daily dosage rates in therange 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250mg. Such effective daily doses will, however, vary depending on inherentactivity of the active ingredient and on the bodyweight of the patient,such variations being within the skill and judgement of the physician.

Cell Cycling

The compounds of the present invention may be useful in the method oftreatment of a cell cycling disorder.

As discussed in “Molecular Cell Biology” 3rd Ed. Lodish et al. pages177-181 different eukaryotic cells can grow and divide at quitedifferent rates. Yeast cells, for example, can divide every 120 min.,and the first divisions of fertilised eggs in the embryonic cells of seaurchins and insects take only 1530 min. because one large pre-existingcell is subdivided. However, most growing plant and animal cells take10-20 hours to double in number, and some duplicate at a much slowerrate. Many cells in adults, such as nerve cells and striated musclecells, do not divide at all; others, like the fibroblasts that assist inhealing wounds, grow on demand but are otherwise quiescent.

Still, every eukaryotic cell that divides must be ready to donate equalgenetic material to two daughter cells. DNA synthesis in eukaryotes doesnot occur throughout the cell division cycle but is restricted to a partof it before cell division.

The relationship between eukaryotic DNA synthesis and cell division hasbeen thoroughly analysed in cultures of mammalian cells that were allcapable of growth and division. In contrast to bacteria, it was found,eukaryotic cells spend only a part of their time in DNA synthesis, andit is completed hours before cell division (mitosis). Thus a gap of timeoccurs after DNA synthesis and before cell division; another gap wasfound to occur after division and before the next round of DNAsynthesis. This analysis led to the conclusion that the eukaryotic cellcycle consists of an M (mitotic) phase, a G₁ phase (the first gap), theS (DNA synthesis) phase, a G₂ phase (the second gap), and back to M. Thephases between mitoses (G₁, S, and G₂) are known collectively as theinterphase.

Many nondividing cells in tissues (for example, all quiescentfibroblasts) suspend the cycle after mitosis and just prior to DNAsynthesis; such “resting” cells are said to have exited from the cellcycle and to be in the G₀ state.

It is possible to identify cells when they are in one of the threeinterphase stages of the cell cycle, by using a fluorescence-activatedcell sorter (FACS) to measure their relative DNA content: a cell that isin G₁ (before DNA synthesis) has a defined amount x of DNA; during S(DNA replication), it has between x and 2x; and when in G₂ (or M), ithas 2x of DNA.

The stages of mitosis and cytokinesis in an animal cell are as follows

(a) Interphase. The G₂ stage of interphase immediately precedes thebeginning of mitosis. Chromosomal DNA has been replicated and bound toprotein during the S phase, but chromosomes are not yet seen as distinctstructures. The nucleolus is the only nuclear substructure that isvisible under light microscope. In a diploid cell before DNA replicationthere are two morphologic chromosomes of each type, and the cell is saidto be 2n. In G₂, after DNA replication, the cell is 4n. There are fourcopies of each chromosomal DNA. Since the sister chromosomes have notyet separated from each other, they are called sister chromatids.

b) Early prophase. Centrioles, each with a newly formed daughtercentriole, begin moving toward opposite poles of the cell; thechromosomes can be seen as long threads. The nuclear membrane begins todisaggregate into small vesicles.

(c) Middle and late prophase. Chromosome condensation is completed; eachvisible chromosome structure is composed of two chromatids held togetherat their centromeres. Each chromatid contains one of the two newlyreplicated daughter DNA molecules. The microtubular spindle begins toradiate from the regions just adjacent to the centrioles, which aremoving closer to their poles. Some spindle fibres reach from pole topole; most go to chromatids and attach at kinetochores.

(d) Metaphase. The chromosomes move toward the equator of the cell,where they become aligned in the equatorial plane. The sister chromatidshave not yet separated.

(e) Anaphase. The two sister chromatids separate into independentchromosomes. Each contains a centromere that is linked by a spindlefibre to one pole, to which it moves. Thus one copy of each chromosomeis donated to each daughter cell. Simultaneously, the cell elongates, asdo the pole-to-pole spindles. Cytokinesis begins as the cleavage furrowstarts to form.

(f) Telophase. New membranes form around the daughter nuclei; thechromosomes uncoil and become less distinct, the nucleolus becomesvisible again, and the nuclear membrane forms around each daughternucleus. Cytokinesis is nearly complete, and the spindle disappears asthe microtubules and other fibres depolymerise. Throughout mitosis the“daughter” centriole at each pole grows until it is full-length. Attelophase the duplication of each of the original centrioles iscompleted, and new daughter centrioles will be generated during the nextinterphase.

(g) Interphase. Upon the completion of cytokinesis, the cell enters theG₁ phase of the cell cycle and proceeds again around the cycle.

It will be appreciated that cell cycling is an extremely important cellprocess. Deviations from normal cell cycling can result in a number ofmedical disorders. Increased and/or unrestricted cell cycling may resultin cancer. Reduced cell cycling may result in degenerative conditions.Use of the compound of the present invention may provide a means totreat such disorders and conditions.

Thus, the compound of the present invention may be suitable for use inthe treatment of cell cycling disorders such as cancers, includinghormone dependent and hormone independent cancers.

In addition, the compound of the present invention may be suitable forthe treatment of cancers such as breast cancer, ovarian cancer,endometrial cancer, sarcomas, melanomas, prostate cancer, pancreaticcancer etc. and other solid tumours.

For some applications, cell cycling is inhibited and/or prevented and/orarrested, preferably wherein cell cycling is prevented and/or arrested.In one aspect cell cycling may be inhibited and/or prevented and/orarrested in the G₂/M phase. In one aspect cell cycling may beirreversibly prevented and/or inhibited and/or arrested, preferablywherein cell cycling is irreversibly prevented and/or arrested.

By the term “irreversibly prevented and/or inhibited and/or arrested” itis meant after application of a compound of the present invention, onremoval of the compound the effects of the compound, namely preventionand/or inhibition and/or arrest of cell cycling, are still observable.More particularly by the term “irreversibly prevented and/or inhibitedand/or arrested” it is meant that when assayed in accordance with thecell cycling assay protocol presented herein, cells treated with acompound of interest show less growth after Stage 2 of the protocol Ithan control cells. Details on this protocol are presented below.

Thus, the present invention provides compounds which: cause inhibitionof growth of oestrogen receptor positive (ER+) and ER negative (ER−)breast cancer cells in vitro by preventing and/or inhibiting and/orarresting cell cycling; and/or cause regression of nitroso-methyl urea(NMU)-induced mammary tumours in intact animals (i.e. notovariectomised), and/or prevent and/or inhibit and/or arrest cellcycling in cancer cells; and/or act in vivo by preventing and/orinhibiting and/or arresting cell cycling and/or act as a cell cyclingagonist.

Cell Cycling Assay Protocol 7

Procedure

Stage 1

MCF-7 breast cancer cells are seeded into multi-well culture plates at adensity of 105 cells/well. Cells were allowed to attach and grown untilabout 30% confluent when they are treated as follows:

Control—No Treatment

Compound of Interest (COI) 20 μM

Cells are grown for 6 days in growth medium containing the COI withchanges of medium/COI every 3 days. At the end of this period cellnumbers were counted using a Coulter cell counter.

Stage 2

After treatment of cells for a 6-day period with the COI cells arere-seeded at a density of 10⁴ cells/well. No further treatments areadded. Cells are allowed to continue to grow for a further 6 days in thepresence of growth medium. At the end of this period cell numbers areagain counted.

Cancer

As indicated, the compounds of the present invention may be useful inthe treatment of a cell cycling disorder. A particular cell cyclingdisorder is cancer.

Cancer remains a major cause of mortality in most Western countries.Cancer therapies developed so far have included blocking the action orsynthesis of hormones to inhibit the growth of hormone-dependenttumours. However, more aggressive chemotherapy is currently employed forthe treatment of hormone-independent tumours.

Hence, the development of a pharmaceutical for anti-cancer treatment ofhormone dependent and/or hormone independent tumours, yet lacking someor all of the side-effects associated with chemotherapy, would representa major therapeutic advance.

It is known that oestrogens undergo a number of hydroxylation andconjugation reactions after their synthesis. Until recently it wasthought that such reactions were part of a metabolic process thatultimately rendered oestrogens water soluble and enhanced theirelimination from the body. It is now evident that some hydroxymetabolites (e.g. 2-hydroxy and 16alpha-hydroxy) and conjugates (e.g.oestrone sulphate, E1S) are important in determining some of the complexactions that oestrogens have in the body.

Workers have investigated the formation of 2- and 16-hydroxylatedoestrogens in relation to conditions that alter the risk of breastcancer. There is now evidence that factors which increase 2-hydroxylaseactivity are associated with a reduced cancer risk, while thoseincreasing 16alpha-hydroxylation may enhance the risk of breast cancer.Further interest in the biological role of estrogen metabolites has beenstimulated by the growing body of evidence that 2-methoxyoestradiol isan endogenous metabolite with anti-mitotic properties. 2-MeOE2 is formedfrom 2-hydroxy estradiol (2-OHE2) by catechol estrogen methyltransferase, an enzyme that is widely distributed throughout the body.

Workers have shown that in vivo 2-MeOE2 inhibits the growth of tumoursarising from the subcutaneous injection of Meth A sarcoma, B16 melanomaor MDA-MB-435 estrogen receptor negative (ER−) breast cancer cells. Italso inhibits endothelial cell proliferation and migration, and in vitroangiogenesis. It was suggested that the ability of 2-MeOE2 to inhibittumour growth in vivo may be due to its ability to inhibittumour-induced angiogenesis rather than direct inhibition of theproliferation of tumour cells.

The mechanism by which 2-MeOE2 exerts its potent anti-mitogenic andanti-angiogenic effects is still being elucidated. There is evidencethat at high concentrations it can inhibit microtubule polymerisationand act as a weak inhibitor of colchicine binding to tubulin. Recently,however, at concentrations that block mitosis, tubulin filaments incells were not found to be depolymerised but to have an identicalmorphology to that seen after taxol treatment. It is possible,therefore, that like taxol, a drug that is used for breast and ovarianbreast cancer therapy, 2-MeOE2 acts by stabilising microtubule dynamics.

While the identification of 2-MeOE2 as a new therapy for cancerrepresents an important advance, the bioavailability of orallyadministered oestrogens is poor. Furthermore, they can undergo extensivemetabolism during their first pass through the liver. As part of aresearch programme to develop a steroid sulphatase inhibitor for breastcancer therapy, oestrone-3-O-sulphamate (EMATE) was identified as apotent active site-directed inhibitor. Unexpectedly, EMATE proved topossess potent oestrogenic properties with its oral uterotrophicactivity in rats being a 100-times higher than that of estradiol. Itsenhanced oestrogenicity is thought to result from its absorption by redblood cells (rbcs) which protects it from inactivation during itspassage through the liver and which act as a reservoir for its slowrelease for a prolonged period of time. A number of A-ring modifiedanalogues were synthesised and tested, including2-methoxyoestrone-3-O-sulphamate. While this compound was equipotentwith EMATE as a steroid sulphatase inhibitor, it was devoid ofoestrogenicity.

We believe that the compound of the present invention provides a meansfor the treatment of cancers and, especially, breast cancer.

In addition or in the alternative the compound of the present inventionmay be useful in the blocking the growth of cancers including leukaemiasand solid tumours such as breast, endometrium, prostate, ovary andpancreatic tumours.

Therapy Concerning Oestrogen

We believe that some of the compounds of the present invention may beuseful in the control of oestrogen levels in the body—in particular infemales. Thus, some of the compounds may be useful as providing a meansof fertility control—such as an oral contraceptive tablet, pill,solution or lozenge. Alternatively, the compound could be in the form ofan implant or as a patch.

Thus, the compounds of the present invention may be useful in treatinghormonal conditions associated with oestrogen.

In addition or in the alternative the compound of the present inventionmay be useful in treating hormonal conditions in addition to thoseassociated with oestrogen. Hence, the compound of the present inventionmay also be capable of affecting hormonal activity and may also becapable of affecting an immune response.

Neurodegenerative Diseases

We believe that some of the compounds of the present invention may beuseful in the treatment of neurodegenerative diseases, and similarconditions.

By way of example, it is believed that STS inhibitors may be useful inthe enhancing the memory function of patients suffering from illnessessuch as amnesia, head injuries, Alzheimer's disease, epileptic dementia,presenile dementia, post traumatic dementia, senile dementia, vasculardementia and post-stroke dementia or individuals otherwise seekingmemory enhancement.

TH1

We believe that some of the compounds of the present invention may beuseful in TH1 implications.

By way of example, it is believed that the presence of STS inhibitorswithin the macrophage or other antigen presenting cells may lead to adecreased ability of sensitised T cells to mount a TH1 (high IL-2, IFNγlow IL-4) response. The normal regulatory influence of other steroidssuch as glucocorticoids would therefore predominate.

Inflamatory Conditions

We believe that some of the compounds of the present invention may beuseful in treating inflammatory conditions—such as conditions associatedwith any one or more of: autoimmunity, including for example, rheumatoidarthritis, type I and II diabetes, systemic lupus erythematosus,multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis,ulcerative colitis and Crohn's disease, skin disorders e.g. psoriasisand contact dermatitis; graft versus host disease; eczema; asthma andorgan rejection following transplantation.

By way of example, it is believed that STS inhibitors may prevent thenormal physiological effect of DHEA or related steroids on immune and/orinflammatory responses.

The compounds of the present invention may be useful in the manufactureof a medicament for revealing an endogenous glucocorticoid-like effect.

Other Therapies

It is also to be understood that the compound/composition of the presentinvention may have other important medical implications.

For example, the compound or composition of the present invention may beuseful in the treatment of the disorders listed in WO-A-99/52890—viz:

In addition, or in the alternative, the compound or composition of thepresent invention may be useful in the treatment of the disorders listedin WO-A-98/05635. For ease of reference, part of that list is nowprovided: cancer, inflammation or inflammatory disease, dermatologicaldisorders, fever, cardiovascular effects, haemorrhage, coagulation andacute phase response, cachexia, anorexia, acute infection, HIVinfection, shock states, graft-versus-host reactions, autoimmunedisease, reperfusion injury, meningitis, migraine and aspirin-dependentantithrombosis; tumour growth, invasion and spread, angiogenesis,metastases, malignant, ascites and malignant pleural effusion; cerebralischaemia, ischaemic heart disease, osteoarthritis, rheumatoidarthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration,Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn'sdisease and ulcerative colitis; periodontitis, gingivitis; psoriasis,atopic dermatitis, chronic ulcers, epidermolysis bullosa; cornealulceration, retinopathy and surgical wound healing; rhinitis, allergicconjunctivitis, eczema, anaphylaxis; restenosis, congestive heartfailure, endometriosis, atherosclerosis or endosclerosis.

In addition, or in the alternative, the compound or composition of thepresent invention may be useful in the treatment of disorders listed inWO-A-98/07859. For ease of reference, part of that list is now provided:cytokine and cell proliferation/differentiation activity;immunosuppressant or immunostimulant activity (e.g. for treating immunedeficiency, including infection with human immune deficiency virus;regulation of lymphocyte growth; treating cancer and many autoimmunediseases, and to prevent transplant rejection or induce tumourimmunity); regulation of haematopoiesis, e.g. treatment of myeloid orlymphoid diseases; promoting growth of bone, cartilage, tendon, ligamentand nerve tissue, e.g. for healing wounds, treatment of burns, ulcersand periodontal disease and neurodegeneration; inhibition or activationof follicle-stimulating hormone (modulation of fertility);chemotactic/chemokinetic activity (e.g. for mobilising specific celltypes to sites of injury or infection); haemostatic and thrombolyticactivity (e.g. for treating haemophilia and stroke); antiinflammatoryactivity (for treating e.g. septic shock or Crohn's disease); asantimicrobials; modulators of e.g. metabolism or behaviour; asanalgesics; treating specific deficiency disorders; in treatment of e.g.psoriasis, in human or veterinary medicine.

In addition, or in the alternative, the composition of the presentinvention may be useful in the treatment of disorders listed inWO-A-98/09985. For ease of reference, part of that list is now provided:macrophage inhibitory and/or T cell inhibitory activity and thus,anti-inflammatory activity; anti-immune activity, i.e. inhibitoryeffects against a cellular and/or humoral immune response, including aresponse not associated with inflammation; inhibit the ability ofmacrophages and T cells to adhere to extracellular matrix components andfibronectin, as well as up-regulated fas receptor expression in T cells;inhibit unwanted immune reaction and inflammation including arthritis,including rheumatoid arthritis, inflammation associated withhypersensitivity, allergic reactions, asthma, systemic lupuserythematosus, collagen diseases and other autoimmune diseases,inflammation associated with atherosclerosis, arteriosclerosis,atherosclerotic heart disease, reperfusion injury, cardiac arrest,myocardial infarction, vascular inflammatory disorders, respiratorydistress syndrome or other cardiopulmonary diseases, inflammationassociated with peptic ulcer, ulcerative colitis and other diseases ofthe gastrointestinal tract, hepatic fibrosis, liver cirrhosis or otherhepatic diseases, thyroiditis or other glandular diseases,glomerulonephritis or other renal and urologic diseases, otitis or otherotorhino-laryngological diseases, dermatitis or other dermal diseases,periodontal diseases or other dental diseases, orchitis orepididimo-orchitis, infertility, orchidal trauma or other immune-relatedtesticular diseases, placental dysfunction, placental insufficiency,habitual abortion, eclampsia, pre-eclampsia and other immune and/orinflammatory-related gynaecological diseases, posterior uveitis,intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis,uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitisor cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitispigmentosa, immune and inflammatory components of degenerative fondusdisease, inflammatory components of ocular trauma, ocular inflammationcaused by infection, proliferative vitreo-retinopathies, acute ischaemicoptic neuropathy, excessive scarring, e.g. following glaucoma filtrationoperation, immune and/or inflammation reaction against ocular implantsand other immune and inflammatory-related ophthalmic diseases,inflammation associated with autoimmune diseases or conditions ordisorders where, both in the central nervous system (CNS) or in anyother organ, immune and/or inflammation suppression would be beneficial,Parkinson's disease, complication and/or side effects from treatment ofParkinson's disease, AIDS-related dementia complex HIV-relatedencephalopathy, Devic's disease, Sydenham chorea, Alzheimer's diseaseand other degenerative diseases, conditions or disorders of the CNS,inflammatory components of stokes, post-polio syndrome, immune andinflammatory components of psychiatric disorders, myelitis,encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis,acute neuropathy, subacute neuropathy, chronic neuropathy,Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis,pseudo-tumour cerebri, Down's Syndrome, Huntington's disease,amyotrophic lateral sclerosis, inflammatory components of CNScompression or CNS trauma or infections of the CNS, inflammatorycomponents of muscular atrophies and dystrophies, and immune andinflammatory related diseases, conditions or disorders of the centraland peripheral nervous systems, post-traumatic inflammation, septicshock, infectious diseases, inflammatory complications or side effectsof surgery, bone marrow transplantation or other transplantationcomplications and/or side effects, inflammatory and/or immunecomplications and side effects of gene therapy, e.g. due to infectionwith a viral carrier, or inflammation associated with AIDS, to suppressor inhibit a humoral and/or cellular immune response, to treat orameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia,by reducing the amount of monocytes or lymphocytes, for the preventionand/or treatment of graft rejection in cases of transplantation ofnatural or artificial cells, tissue and organs such as cornea, bonemarrow, organs, lenses, pacemakers, natural or artificial skin tissue.

Compound Preparation

The compounds of the present invention may be prepared by reacting anappropriate alcohol with a suitable chloride. By way of example, thesulphamate compounds of the present invention may be prepared byreacting an appropriate alcohol with a suitable sulfamoyl chloride, ofthe formula R₃R₄NSO₂Cl.

Typical conditions for carrying out the reaction are as follows.

Sodium hydride and a sulfamoyl chloride are added to a stirred solutionof the alcohol in anhydrous dimethyl formamide at 0° C. Subsequently,the reaction is allowed to warm to room temperature whereupon stirringis continued for a further 24 hours. The reaction mixture is poured ontoa cold saturated solution of sodium bicarbonate and the resultingaqueous phase is extracted with dichloromethane. The combined organicextracts are dried over anhydrous MgSO₄. Filtration followed by solventevaporation in vacuo and co-evaporated with toluene affords a cruderesidue which is further purified by flash chromatography.

Preferably, the alcohol is derivatised, as appropriate, prior toreaction with the sulfamoyl chloride. Where necessary, functional groupsin the alcohol may be protected in known manner and the protecting groupor groups removed at the end of the reaction.

Preferably, the sulphamate compounds are prepared according to theteachings of Page et al (1990 Tetrahedron 46; 2059-2068).

The phosphonate compounds may be prepared by suitably combining theteachings of Page et al (1990 Tetrahedron 46; 2059-2068) andPCT/GB92/01586.

The sulphonate compounds may be prepared by suitably adapting theteachings of Page et al (1990 Tetrahedron 46; 2059-2068) andPCT/GB92/01586.

The thiophosphonate compounds may be prepared by suitably adapting theteachings of Page et al (1990 Tetrahedron 46; 2059-2068) andPCT/GB91/00270.

Preferred preparations are also presented in the following text.

SUMMARY

In summation, the present invention provides novel compounds for use assteroid sulphatase inhibitors and/or aromatase inhibitors and/ormodulators of apoptosis and/or modulators of cell cycling and/or cellgrowth, and pharmaceutical compositions containing them.

EXAMPLES

The present invention will now be described in further detail by way ofexample only with reference to the accompanying figure in which:

FIG. 1 shows a summary scheme; and

FIG. 2 shows a summary scheme.

The present invention will now be described only by way of example.However, it is to be understood that the examples also present preferredcompounds of the present invention, as well as preferred routes formaking same and useful intermediates in the preparation of same.

Synthetic Routes

Compounds in accordance with the present invention were synthesised inaccordance with the synthetic routes and schemes.

The present invention will now be described only by way of example.However, it is to be understood that the examples also present preferredcompounds of the present invention, as well as preferred routes formaking same and useful intermediates in the preparation of same.

Synthetic Routes

Compounds in accordance with the present invention were synthesised inaccordance with the synthetic routes and schemes.

Scheme. Synthesis of Anastrozole

3-Bromomethyl-5-methyl-benzoic acid methyl ester (LWO03036B) and3,5-Bis-bromomethyl-benzoic acid methyl ester (LWO03036C)

To a solution of methyl 3,5-dimethylbenzoate (15.0 g, 89.52 mmol) incarbon tetrachloride (molecular sieves 4 Å dried, 150 mL) was addedfinely powdered N-bromosuccinimide (16.1 g, 89.52 mmol) and benzoylperoxide (≧97%, 200 mg). The light yellow suspension was then refluxedwhich turned into a light orange suspension after about 10 min. When thecolour of the suspension returned to light yellow again about 45 minlater, the reflux was terminated. After cooling to room temperature, thesuspension was filtered and the filter cake collected was washed withether (5×30 mL). The combined filtrates were evaporated to give a clearbright yellow liquid (22.9 g) which was fractionated by flashchromatography (silica: 700 g; eluent: ethyl acetate/hexane, 1:10 to1:1). The second fraction that collected upon evaporation gave LWO03036Bas a translucent pale yellow oil (13.32 g, 54.79 mmol, 61%) whereas thethird fraction gave LWO03036C as white fluffy needle-shaped crystals(1.99 g, 3.08 mmol, 7%).

LWO03036B:

R_(f): 0.27 (ethyl acetate/hexane, 1:8), c.f. 0.39 (S.M.) δ_(H) (270MHz, CDCl₃) 2.39 (3H, s, Ar—CH₃), 3.90 (3H, s, COOMe), 4.47 (2H, s,CH₂Br), 7.39 (1H, slightly broad s, Ar), 7.78 (1H, slightly broad s, Ar)and 7.85 (1H, s, Ar). LRMS (FAB+): 443.3 (17), 398.3 [25,(M⁸¹Br+H+NBA)+], 243.1 [100, (M⁷⁹Br+H)⁺], 163.1 [83, (M⁷⁹Br−⁷⁹Br)⁺],85.1 (54). HRMS (FAB+): 243.00124 C₁₀H₁₂O₂Br requires 243.00207

LWO03036C:

R_(f): 0.20 (ethyl acetate/hexane, 1:8), c.f. 0.39 (S.M.) m.p. 100-103°C. [Lit.¹ (from column), 95-97° C.] ¹ Liu P, Chen Y, Deng J, Tu Y. Anefficient method for the preparation of benzylic bromides. Synthesis2001, 14: 2078-2080. δ_(H) (400 MHz, CDCl₃) 3.93 (3H, s, OCH₃), 4.49(4H, s, 2×CH₂), 7.60 (1H, t, J˜1.7 Hz, C4-H) and 7.97 (2H, d, J˜2.0 Hz,C2-H and C6-H).

3,5-Bis-cyanomethyl-benzoic acid methyl ester (LWO03041A)

A mixture of LWO03036C (1.96 g, 6.081 mmol), LWO03015C (913 mg, 2.835mmol), potassium cyanide (1.45 g, 21.40 mmol), tetrabutylammoniumbromide (100 mg) in dichloromethane (15 mL) and water (5 mL) wasrefluxed with vigorous stirring for 4 h. After removal of volatilesolvent, the concentrated mixture was diluted with ethyl acetate (50mL). The organic layer was washed with brine (100 mL, 4×50 mL), dried(MgSO₄), filtered and evaporated to give a golden yellow syrup which onstanding at room temperature solidified to a yellow mass. This crudeproduct was fractionated by flash chromatography (silica: 80 g; eluent:ethyl acetate/hexane, 1:1) and the fourth fraction that isolated uponevaporation gave LWO03041A as white residue (1.11 g, 5.182 mmol, 58%);R_(f): 0.41 (ethyl acetate/hexane, 1:1), c.f. 0.78 (S.M.) m.p. 91-93° C.[Lit. (Patent: EP0296749A1, 90-92° C. (CCl₄)]; δ_(H) (270 MHz, CDCl₃)3.82 (4H, s, 2×CH₂), 3.95 (3H, s, COOMe), 7.53 (1H, s, ArH) and 7.99(2H, s, 2×ArH).

Found: C 67.1, H 4.74, N 13.0%. C₁₂H₁₀N₂O₂ requires C 67.28, H 4.71, N13.08%.

3,5-Bis-(cyano-dimethyl-methyl)-benzoic acid methyl ester (LWO03043)

To a solution of LWO03041A (1.09 g, 5.088 mmol) in anhydrous DMF (20 mL)at ice/water temperature was added cautiously sodium hydride (60% inmineral oil, 896 mg, 22.39 mmol) in four portions. After stirring atthis temperature under an atmosphere of nitrogen for 15 min, methyliodide (1.39 mL, 22.39 mmol) was introduced and the resulting suspensionwas stirred at room temperature overnight. The light orange suspensionobtained was diluted with ethyl acetate (50 mL) and the organic layerwashed with brine (200 mL, 5×50 mL), dried (MgSO₄), filtered andevaporated to give a yellow/brown syrup. This crude product wasfractionated by flash chromatography (silica: 90 g; eluent: ethylacetate/hexane, 1:1) and the main fraction that isolated uponevaporation gave a bright yellow syrup which on standing at roomtemperature solidified to give LWO03043 as creamy wax (1.33 g, 4.920mmol, 97%). A small amount of this wax (535 mg) was dissolved in hotcyclohexane (˜3 mL). Upon cooling of the mixture a light yellow oil wasinitially separated which solidified to a creamy mass at roomtemperature.

R_(f): 0.65 (ethyl acetate/hexane, 1:1), c.f. 0.46 (S.M.) m.p. 85-87° C.[Lit. (Patent: EP0296749A1, 83-85° C. (CCl₄)]; δ_(H) (270 MHz, CDCl₃)1.77 (12H, s, 4×Me), 3.95 (3H, s, COOMe), 7.80 (1H, t, J˜1.8 Hz, C4-H)and 8.08 (2H, d, J 2 Hz, C2-H and C6-H). Found: C 71.1, H 6.78, N 10.3%.C₁₆H₁₈N₂O₂ requires C 71.09, H 6.71, N 10.36%.

2-[3-(Cyano-dimethyl-methyl)-5-hydroxymethyl-phenyl]-2-methyl-propionitrile(LWO03044, LWO03049A)

To a mixture of LWO03043 (516 mg, 1.909 mmol) and lithium borohydride(88 mg, 3.817 mmol) at room temperature was added anhydrous THF (10 mL).The resulting clear pale yellow solution was refluxed under anatmosphere of nitrogen for 3 h, then cooled to ice/water temperature andtreated dropwise (cautiously) with 1 M hydrochloric acid until themixture remained acidic. Ethyl acetate (100 mL) was added and theorganic layer was washed with brine (100 mL, 4×50 mL), dried (MgSO₄),filtered and evaporated to give LWO03049 as a white residue (460 mg,1.898 mmol, 99.5%).

R_(f): 0.23 (ethyl acetate/hexane, 1:1), c.f. 0.48 (S.M.) m.p. 150-160°C. [Lit. (Patent: EP0296749A1, 151-153° C.]; δ_(H) (270 MHz, CDCl₃) 1.75(12H, s, 4×Me), 4.76 (2H, s, CH₂O), 7.44 (2H, br s, ArH) and 7.46 (1H,d, J 1.7 Hz, ArH). LHMS (FAB+): 501.3 (5), 485.3 [24, (2M+H)⁺], 467.3[35, (2M+H−H₂O)⁺], 396.2 [24, (M+H+NBA)⁺], 378.2 [22, (M+H+NBA−H₂O)⁺],272.1 (8), 247.2 (43), 225.1 [100, (M+H−H₂O)⁺]. LHMS (FAB−): 708.5 (20),619.4 (30), 571.4 (18), 556.5 (25), 495.3 (31), 482.3 (35), 467.3 (27),420.4 (26), 395.4 [100, M+NBA)⁻], 331.2 (43). HRMS (FAB+): 396.19317,C₂₂H₂₆N₃O₄ (M+H+NBA) requires 396.19233. HRMS (FAB+): 225.14394,C₁₅H₁₇N₂ (M+H−H₂O) requires 225.13917.

LWO03049 contained about 3-5% w/w of starting material (the ester) andwas used without further purification.

2-[3-(Cyano-dimethyl-methyl)-5-[1,2,4]triazol-1-ylmethyl-phenyl]-2-methyl-propionitrile(LWO03051A, Anastrozole, STX1034)(c.f. LWO03045)

To a solution of LWO03049 (418 mg, 1.725 mmol) in anhydrousdichloromethane (15 mL) at ice/water temperature under an atmosphere ofnitrogen was added anhydrous pyridine (0.15 mL, 1.897 mmol) followed bythionyl chloride (0.19 mL, 2.588 mmol). The reaction mixture was stirredat ice/water temperature for 15 min and then at room temperature for 2 hbefore subjecting to reflux for 1 h. After cooling, the reaction mixturewas evaporated and the white residue obtained in ethyl acetate (30 mL)was washed with brine (3×30 mL). The organic layer was dried (MgSO₄),filtered and evaporated to give a pale yellow residue (473 mg,LWO03050). To a solution of this residue in anhydrous DMF (4 mL) atice/water temperature was added 1,2,4-triazole, sodium derivative (367mg, 3.628 mmol). The resulting light brown suspension was stirred atroom temperature under an atmosphere of nitrogen for 18 h. Ethyl acetate(30 mL) was added to dilute the reaction mixture and the organic layerwas washed with brine (50 mL, 3×30 mL), dried (MgSO₄), filtered andevaporated to give a light yellow brown syrup (438 mg). This crudeproduct was fractioned by flash chromatography (silica: 15 g; eluent:ethyl acetate) and the second fraction that isolated upon evaporationgave a light yellow glue which upon standing for weeks at roomtemperature solidified to give LWO03051 as creamy pale yellow wax (279mg, 951 μmol, 52%).

R_(f): 0.33 (ethyl acetate), c.f. 0.62 (LWO03049). m.p. 59-62° C. [Lit.(Patent: EP0296749A1, 81-82° C. (ethyl acetate/cyclohexane)]; δ_(H) (270MHz, CDCl₃) 1.71 (12H, s, 4×Me), 5.39 (2H, s, CH₂), 7.32 (2H, two s,C4-H and C6-H), 7.53 (1H, t, J 1.7 Hz, C2-H), 7.80 (1H, s, C3′-H) and8.14 (1H, s, C5′-H). Found: C 69.6, H 6.38, N 23.9%. C₁₇H₁₉N₅ requires C69.60, H 6.53, N 23.87%.

Scheme. Synthesis of Anastrozole-based DASI (STX1023)

3-Bromomethyl-5-methyl-benzoic acid methyl ester (LWO03015B) and3,5-Bis-bromomethyl-benzoic acid methyl ester (LWO03015C)

To a solution of methyl 3,5-dimethylbenzoate (5.57 g, 33.24 mmol) incarbon tetrachloride (molecular sieves 4 Å dried, 50 mL) was addedfinely powdered N-bromosuccinimide (5.98 g, 33.24 mmol) and benzoylperoxide (≧97%, 100 mg). The light yellow suspension was then refluxedwhich turned into a light orange suspension after about one hour. Whenthe colour of the suspension returned to light yellow again about 30 minlater, the reflux was terminated. After cooling to room temperature, thesuspension was filtered and the filter cake collected was washed withether (5×30 mL). The combined filtrates were evaporated to give a clearbright yellow liquid (8.15 g) which was fractionated by flashchromatography (silica: 300 g; eluent: ethyl acetate/hexane, 1:10; flowrate: ca. 30 mL/min). The first fraction collected was the startingmaterial (998 mg). The second and third fractions collected wererespectively LWO03015B as a translucent pale yellow oil (4.90 g, 20.16mmol, 61%) and LWO03015C as white fluffy needle-shaped crystals (990 mg,3.08 mmol, 9%).

LWO03015B

R_(f): 0.27 (ethyl acetate/hexane, 1:8), c.f. 0.39 (S.M.) δ_(H) (270MHz, CDCl₃) 2.39 (3H, s, Ar—CH₃), 3.90 (3H, s, COOMe), 4.47 (2H, s,CH₂Br), 7.39 (1H, slightly broad s, Ar), 7.78 (1H, slightly broad s, Ar)and 7.85 (1H, s, Ar). LRMS (FAB+): 443.3 (17), 398.3 [25,(M⁸¹Br+H+NBA)⁺], 243.1 [100, (M⁷⁹Br+H)⁺], 163.1 [83, (M⁷⁹Br−⁷⁹Br)⁺],85.1 (54). HRMS (FAB+): 243.00124 C₁₀H₁₂O₂Br requires 243.00207.

LWO03015C

R_(f): 0.20 (ethyl acetate/hexane, 1:8), c.f. 0.39 (S.M.) m.p. 100-103°C. [Lit.¹ (from column), 95-97° C.] ¹ Liu P, Chen Y, Deng J, Tu Y. Anefficient method for the preparation of benzylic bromides. Synthesis2001, 14: 2078-2080. δ_(H) (400 MHz, CDCl₃) 3.93 (3H, s, OCH₃), 4.49(4H, s, 2×CH₂), 7.60 (1H, t, J˜1.7 Hz, C4-H) and 7.97 (2H, d, J˜2.0 Hz,C2-H and C6-H).

3-Cyanomethyl-5-methyl-benzoic acid methyl ester (LWO03016B)

A mixture of LWO03015B (3.0 g, 12.34 mmol), potassium cyanide (1.0 g,14.81 mmol), tetrabutylammonium bromide (100 mg) in dichloromethane (15mL) and water (5 mL) was refluxed with vigorous stirring for 4 h. Afterremoval of volatile solvent, the concentrated mixture was diluted withethyl acetate (100 mL). The organic layer was washed with brine (100 mL,4×50 mL), dried (MgSO₄), filtered and evaporated to give a lightyellow/brown syrup which upon standing at room temperature solidified toa yellow mass (2.48 g). This crude product was fractionated by flashchromatography (silica: 100 g; eluent: ethyl acetate/hexane, 1:4, then1:2 after collection of the first fraction). The second fraction thatisolated upon evaporation gave LWO03016B as an almost colourless syrupwhich upon standing at room temperature solidified into a mass of whitewax (1.84 g, 9.725 mmol, 79%); R_(f): 0.20 (ethyl acetate/hexane, 1:4),c.f. 0.44 (S.M.); m.p. 55-57° C.; δ_(H) (270 MHz, CDCl₃) 2.41 (3H, s,CH₃), 3.75 (2H, s, CH₂), 3.93 (3H, s, OCH₃), 7.35 (1H, s, ArH), 7.78(1H, s, ArH) and 7.82 (1H, s, ArH).

LRMS (FAB+): 496.1 [6, (M+H+2NBA)⁺], 343.1 [32, (M+H+NBA)⁺], 190.1 [100,(M+H)⁺], 158.1 [22, (M−OMe)⁺]. HRMS (FAB+): 190.08651 C₁₁H₁₂NO₂ requires190.08680. Found: C 69.4, H 5.87, N 7.19%. C₁₁H₁₁NO₂ requires C 69.83, H5.86, N 7.40%.

3-(Cyano-dimethyl-methyl)-5-methyl-benzoic acid methyl ester (LWO03017A)

To a solution of LWO03016B (1.72 g, 9.075 mmol) in anhydrous DMF (20 mL)at ice/water temperature was added cautiously sodium hydride (60% inmineral oil, 800 mg, 19.96 mmol) in four portions. After stirring atthis temperature under an atmosphere of nitrogen for 15 min, methyliodide (2.83 g, 19.96 mmol) was introduced and the resulting suspensionwas stirred at room temperature overnight. The light orange suspensionobtained was diluted with ethyl acetate (100 mL) and the organic layerwashed with brine (200 mL, 4×50 mL), dried (MgSO₄), filtered andevaporated to give a light orange/brown syrup (2.30 g). This crudeproduct was fractionated by flash chromatography (silica: 90 g; eluent:ethyl acetate/hexane, 1:4) and the first fraction that isolated uponevaporation gave a pale yellow syrup which upon standing at roomtemperature solidified to give LWO03017A as a mass of white wax (1.74 g,8.009 mmol, 88%).

R_(f): 0.32 (ethyl acetate/hexane, 1:4), c.f. 0.21 (S.M.) m.p. 51.5-55°C.; δ_(H) (270 MHz, CDCl₃) 1.73 (6H, s, 2×Me), 2.42 (3H, s, Ar—CH₃),3.91 (3H, s, COOMe), 7.52 (1H, s, ArH), 7.80 (1H, s, ArH) and 7.88 (1H,s, ArH). Found: C 72.1, H 6.98, N 6.48%. C₁₃H₁₅NO₂ requires C 71.87, H6.96, N 6.45%.

3-Bromomethyl-5-(cyano-dimethyl-methyl)-benzoic acid methyl ester(LWO03023C and LWO03025)

To a solution of LWO03017A (1.68 g, 7.733 mmol) in carbon tetrachloride(molecular sieves 4 Å dried, 20 mL) was added finely powderedN-bromosuccinimide (1.39 g, 7.733 mmol) and benzoyl peroxide (≧97%, 30mg). The light yellow suspension was then refluxed which turned into alight orange suspension after about one hour. When the colour of thesuspension returned to light yellow again about 1 h later, the reactionmixture was cooled to room temperature, filtered and the filter cakecollected washed with ether (5×30 mL). The combined filtrates wereevaporated to give a clear bright yellow syrup which was fractionated byflash chromatography (silica: 250 g; eluent: ethyl acetate/hexane, 1:6to 1:4). The third collected upon evaporation gave a pale yellow syrup(LWO03023C, 1.79 g) which, according to ¹H NMR, contained about 33% of3-dibromomethyl-5-(cyano-dimethyl-methyl)-benzoic acid methyl ester inaddition to the product. δ_(H) (270 MHz, CDCl₃) 1.75 (6H, s, 2×Me), 1.77(equiv of 2H, s), 3.93 (3H, s, COOMe), 3.95 (equiv of 1H), 4.51 (2H, s,CH₂Br), 6.67 (equiv of 0.5H, CHBr₂), 7.72 (1H, t, J<2 Hz, ArH), 7.87(equiv of 0.5H), 8.02 (2H, broad s, 2×ArH), 8.07 (equiv of 0.5H, t, J<2Hz) and 8.20 (equiv of 0.5H, vague t).

To a solution of LWO03023C (261 mg) in anhydrous THF (5 mL) at ice/watertemperature under an atmosphere of nitrogen was added diethyl phosphite(243 mg, 1.762 mmol) and N-ethyl diisopropylamine (228 mg, 1.762 mmol).After stirring at room temperature for 14 h, the reaction mixture waspoured onto ice and extracted with ethyl acetate (30 mL). The organicextract was further washed with 1M hydrochloric acid (50 mL) and thenbrine (3×50 mL), dried (MgSO₄) and evaporated to give LWO03025 as a paleyellow translucent syrup (285 mg, 962 μmol, 85%); R_(f): 0.21 (ethylacetate/hexane, 1:4), c.f. 0.28 (S.M.) δ_(H) (270 MHz, CDCl₃) 1.75 (6H,s, 2×Me), 3.93 (3H, s, COOMe), 4.51 (2H, s, CH₂Br), 7.72 (1H, t, J<2 Hz,ArH) and 8.02 (2H, broad s, 2×ArH).

LWO03025 still contained trace amount of the dibromide impurity but itwas used without further purification.

3-Bromomethyl-5-(cyano-dimethyl-methyl)-benzoic acid methyl ester(LWO03028, c.f. LWO03025)

To a solution of LWO03023C (1.52 g) in anhydrous THF (30 mL) atice/water temperature under an atmosphere of nitrogen was added diethylphosphite (2.9 g, 20.57 mmol) and N-ethyl diisopropylamine (2.69 g,20.57 mmol). After stirring at room temperature for 24 h, the reactionmixture was poured onto ice and extracted with ethyl acetate (50 mL).The organic extract was further washed with 1M hydrochloric acid (100mL) and then brine (4×50 mL), dried (MgSO₄) and evaporated to giveLWO03028 as a pale yellow translucent syrup (1.52 g, 5.132 mmol, 78%);R_(f): 0.21 (ethyl acetate/hexane, 1:4), c.f. 0.28 (S.M.) δ_(H) (270MHz, CDCl₃) 1.77 (6H, s, 2×Me), 3.94 (3H, s, COOMe), 4.52 (2H, s,CH₂Br), 7.71 (1H, t, J˜1.7 Hz, ArH) and 8.02 (2H, m, 2×ArH).

3-(Cyano-dimethyl-methyl)-5-[1,2,4]triazol -1-ylmethyl-benzoic acidmethyl ester (LWO03035)

To a solution of LWO03028 (986 mg, 3.329 mmol) in anhydrous DMF (5 mL)at ice/water temperature was added 1,2,4-triazole, sodium derivative(1.35 g, 13.32 mmol). Under an atmosphere of nitrogen, the resultingsuspension was stirred at room temperature for 18 h before it wasdiluted with ethyl acetate (30 mL). The organic layer was washed withbrine (70 mL, 4×30 mL), dried (MgSO₄), filtered and evaporated to give alight yellow syrup (860 mg). This crude product was then purified byflash chromatography (silica: 50 g, eluent: ethyl acetate). The secondfraction that collected gave a clear pale yellow glue (LWO03035, 667 mg,2.346 mmol, 70%) which upon standing at room temperature for 15 minturned into a mass of white wax; δ_(H) (270 MHz, CDCl₃) 1.73(6H, s,2×Me), 3.92 (3H, s, COOMe), 5.41 (2H, s, CH₂N), 7.61 (1H, t, ArH), 7.86(1H, s, ArH), 7.99 (1H, s, C3′-H), 8.08 (1H, t, J 1.5 Hz, ArH) and 8.14(1H, s, C5′-H).

2-(3-Hydroxymethyl-5-[1,2,4]triazol-1-ylmethyl-phenyl)-2-methyl-propionitrile(LWO03037)

A mixture of LWO03035 (668 mg, 2.350 mmol), sodium borohydride powder(272 mg, 7.049 mmol) and PEG 400 (˜8 g) was heated at 80° C. withstirring and swirling of flask for 18 h. After cooling, the syrup/gluewas diluted and sonicated with ethyl acetate (30 mL). The organic layerwas washed with 1M HCl (50 mL), basified with saturated sodiumbicarbonate solution and then washed with brine (4×30 mL), dried(MgSO₄), filtered and evaporated to give LWO03037 as a light yellowresidue (335 mg, 1.307 mmol, 56%).

R_(f): 0.14 (ethyl acetate), c.f. 0.21 (S.M.); δ_(H) (270 MHz, CDCl₃)1.70 (6H, s, 2×Me), 1.97 (1H, t, J 5.5 Hz, OH), 4.71 (2H, d, J 5.4 Hz,CH₂O), 5.36 (2H, s, CH₂N), 7.19 (1H, s, ArH), 7.30 (1H, s, ArH), 7.45(1H, s, ArH), 7.96 (1H, s, C3′-H) and 8.09 (1H, s, C5′-H).

2-[3-(4-Hydroxy-phenylsulfanylmethyl)-5-[1,2,4]triazol-1-ylmethyl-phenyl]-propionitrile[LWO03038→LWO03039(STX1022)]

To a solution of LWO03037 (322 mg, 1.256 mmol) in anhydrousdichloromethane (15 mL) at ice/water temperature under an atmosphere ofnitrogen was added anhydrous pyridine (0.15 mL, 1.884 mmol) followed bythionyl chloride (0.14 mL, 1.884 mmol). The reaction mixture was stirredat room temperature for 2 h and then refluxed for 1 h. After cooling andremoval of volatile solvent, the brown oil that obtained in anhydrousDMF (5 mL) at room temperature and under an atmosphere of nitrogen wastreated with finely-powdered potassium carbonate (1.74 g, 12.56 mmol)followed by a solution of 4-hydroxythiophenol (196 mg, 1.507 mmol) inanhydrous DMF (0.5 mL). The resulting yellow brown suspension was heatedat 50° C. for 18 h and then diluted with ethyl acetate (40 mL). Theorganic layer was washed with brine (100 mL, 4×30 mL), dried (MgSO₄),filtered and evaporated to give a dark brown syrup (LWO03038, 635 mg).This crude product was fractionated by flash chromatography (silica: 70g; eluent: ethyl acetate). The fourth fraction that isolated gaveLWO03039 as a light yellow brown syrup (120 mg) which was furtherpurified by eluting through an Isolute column (5 g) with ethyl acetateto give LWO03039B (90 mg, 247 μmol, 20%); R_(f): 0.38 (ethyl acetate),c.f. 0.38 (LWO03038); δ_(H) (400 MHz, CDCl₃) 1.66 (6H, s, 2×Me), 3.87(2H, s, CH₂S), 5.25 (2H, s, CH₂N), 6.68 (2H, AA′BB′), 6.74 (1H, s, ArH),7.04 (2H, AA′BB′), 7.19 (1H, t, ArH), 7.24 (1H, t, ArH), 7.95 (1H, s,C3′-H) and 7.99 (1H, s, C5′-H) and 8.70 (1H, br s, OH).

LCMS (ES+): 365.1 [100, (M+H)⁺], 296.0 [7, (M-triazole)⁺], 269 (8);t_(R)=2.34 min. HRMS (FAB+): 365.14410 C₂₀H₂₁N₄OS requires 365.14361.

2-[3-(4-Hydroxy-phenylsulfanylmethyl)-5-[1,2,4]triazol-1-ylmethyl-phenyl]-2-methyl-propionitrile(LWO03042, STX1023)

To a solution of LWO03039B (85 mg, 233.2 μmol) in anhydrous DMA (2 mL)at ice/water temperature was added a concentrated solution of sulphamoylchloride in toluene (2 equiv). The resulting mixture was stirred underan atmosphere of nitrogen at room temperature for 18 h. After dilutingwith ethyl acetate (30 mL), the organic layer was washed with brine (70mL, 5×25 mL), dried (MgSO₄), filtered and evaporated to give a lightyellow brown glue (109 mg). This crude product was fractionated by flashchromatography (silica: 35 g, eluent: ethyl acetate) and the thirdfraction that isolated gave LWO03042 as a pale yellow glue (60 mg, 135.3μmol, 58%).

R_(f): 0.45 (ethyl acetate), c.f. 0.35(S.M.); δ_(H) (400 MHz, CDCl₃)1.73 (6H, s, 2×Me), 4.04 (2H, s, CH₂S), 5.25 (2H, s, CH₂N), 6.58 (1H, s,ArH), 6.87 (2H, s, OSO₂NH₂), 7.15 (4H, s, ArH×4), 7.23 (1H, s, ArH),7.34 (1H, t, ArH), 7.57 (1H, s, C3′-H) and 7.85 (1H, s, C5′-H). LCMS(ES+): 444.26 [100, (M+H)⁺]; t_(R)=2.35 min. HRMS (FAB+): 444.11707.C₂₀H₂₂N₅O₃S₂ requires 444.11641.Synthesis of STX1528

CAB04079

3-Chlorocarbonyl-5-nitro-benzoic acid methyl ester (CAB04079): Asuspension of 5-nitro-isophtalic acid mono methyl ester (22.52 g, 100.0mmol) in thionyl chloride (50 ml) was heated to reflux for 6 hours(until the production of SO₂ and HCl-gas ceased). The excess of thionylchloride was removed under reduced pressure, the residue was dissolvedin DCM and the product was precipitated by addition of hexane to give awhite solid. Yield: 24.10 g (99%).

¹H-NMR (400 MHz, CDCl₃) δ 4.08 (3H, s, —OCH₃), 9.07 (1H, dd, J=1.7, 1.7Hz), 9.12 (1H, dd, J=2.2, 1.7 Hz), 9.16 (1H, dd, 2.2, 1.7 Hz); ¹³C-NMR(100.5 MHz, CDCl₃) δ 53.4 (OCH₃), 129.2, 130.0, 133.2, 135.4, 136.8,148.7, 163.5, 166.1; MS (FAB+): m/z 242.9 (100%, [C₉H₆ClNO₅]⁺)CAB04080

3-Hydroxymethyl-5-nitro-benzoic acid methyl ester (CAB04080): Sodiumborohydride (1.892 g, 50.0 mmol) was added in small portions to EtOH(150 ml) at 0° C. (ice bath), then CAB04079 (12.18 g, 50.0 mmol) wasadded in small portions to the mixture. The solution turned dark redimmediately and was stirred for 1 hour after the addition was complete.The reaction mixture was poured into crushed ice (ca. 300 g) and thecrude product was extracted with EtOAc (3×100 ml). The organic layer waswashed with water (100 ml) and brine (50 ml), dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography (EtOAc/hexane 1:3, R_(f): 0.31) to give the titlecompound as a white solid.

Yield: 4.57 g (43%). ¹H-NMR (400 MHz, CDCl₃) δ 2.17 (1H, br s, —OH),4.02 (3H, s, —OCH₃), 4.92 (2H, s, —CH ₂OH), 8.39 (1H, m), 8.47 (1H, m),8.79 (1H, m); ¹³C-NMR (100.5 MHz, CDCl₃) δ 52.9 (OCH₃), 63.6, 123.5,125.3, 132.0, 133.1 143.6, 165.0 (one carbon not resolved); MS (FAB+):m/z 211.9 (100%, [C₉H₉NO₅]⁺); HPLC (ACS80) t_(r)=1.921 min (>99%).CAB04081

3-Chloromethyl-5-nitro-benzoic acid methyl ester (CAB04081): Thionylchloride (5 ml) was added to a solution of CAB04080 (4.223, 20.0 mmol)in DCM. The dark solution was heated to reflux until the production gasceased (ca. 30 min) and concentrated under reduced pressure. The residuewas purified by column chromatography (EtOAc/hexane 1:5, R_(f): 0.45) togive the title compound as a light yellow solid. Yield: 4.225 g (92%).¹H-NMR (400 MHz, CDCl₃) δ 4.03 (3H, s, —OCH₃), 4.73 (2H, s, —CH₂Cl),8.43 (1H, m), 8.49 (1H, dd, J=2.0, 2.0 Hz), 8.85 (1H, dd, J=2.0, 2.0Hz); ¹³C-NMR (100.5 MHz, CDCl₃) δ 44.1, 53.0 (OCH₃), 124.4, 127.3,132.4, 135.1 140.6, 164.5 (one carbon not resolved); MS (FAB+): m/z229.9 (100%, [C₉H8ClNO₄]⁺); HPLC (ACS80) t_(r)=2.199 min (>99%).STX1313 (CAB04082)

3-Nitro-5-[1,2,4]triazol-1-ylmethyl-benzoic acid methyl ester (CAB04082,STX1313): A mixture of CAB04081 (4.00 g, 17.4 mmol), 1,2,4-triazole(2.40 g, 34.8 mmol), K₂CO₃ (6.91 g, and sodium iodide (0.20 g, 1.33mmol) in acetone (30 ml) was stirred vigorously for 24 hours at roomtemperature. EtOAc (100 ml) and water (50 ml) were added, the organicwas separated, washed with water (50 ml) and brine (30 ml), dried overNa₂SO₄ and concentrated pressure. The residue was purified by columnchromatography (EtOAc, R_(f): 0.36) to give the title compound as alight yellow solid. Yield: 3.878 g (85%).

¹H-NMR (400 MHz, CDCl₃) δ 4.01 (3H, s, —OCH₃), 5.54 (2H, s, —CH₂—), 8.05(1H, s), 8.27 (1H, s), 8.29 (1H, m), 8.35 (1H, dd, J=2.3, 1.8 Hz), 8.85(1H, dd, J=2.0, 1.8 Hz); ¹³C-NMR (100.5 MHz, CDCl₃) δ 52.1, 53.1, 124.7,126.7, 132.7, 134.4, 137.5, 143.5, 148.7, 153.0, 164.4; MS (FAB+): m/z263.0 (100%, [C₁₁H₁₁N₄O₄]⁺); HRMS (FAB+) calcd for C₁₁H₁₁N₄O₄:263.07803; found, 263.07846. HPLC (ACS90) t_(r)=1.843 min (>99%).STX1314 (CAB04083)

3-Amino-5-[1,2,4]triazol-1-ylmethyl-benzoic acid methyl ester (CAB04083,STX1314): Pd/C (100 mg, 5% Pd) was added to a solution of CAB04082(1.311 g, 5.0 mmol) in EtOH (20 ml) and THF (20 ml). The resultingmixture was stirred under H₂-atmosphere for 24 hours. The Pd/C wasremoved by filtration through celite and the resulting clear solutionwas concentrated under reduced pressure. The residue was purified bycolumn chromatography (EtOAc, R_(f): 0.23) to give the title compound asa light yellow solid. Yield: 0.372 g (32%).

₁H-NMR (400 MHz, DMSO-d₆) δ 3.80 (3H, s, —OCH₃), 5.34 (2H, s, —CH₂—),5.52 (2H, s, —NH₂), 6.66 (1 h, dd, J=2.0, 2.0 Hz), 7.01 (1H, dd, J=2.0,2.0 Hz), 7.13 (1H, dd, J=2.0, 2.0 Hz), 8.01 (1H, s), 8.67 (1H,s);¹³C-NMR (100.5 MHz, DMSO-d₆) δ 52.3, 52.4, 114.2, 115.9, 117.6, 131.0,138.0, 144.8, 149.8, 152.2, 166.9; MS (FAB+): m/z 233.0 (100%,[C₁₁H₁₃N₄O₂]⁺); HRMS (FAB+) calcd for C₁₁H₁₃N₄O₂: 233.10385; found,233.10318. HPLC (ACS90) t_(r)=1.771 min (>99%).STX1514 (CAB04121)

3-(4-Benzyloxy-3-fluoro-benzenesulfonylamino)-5-[1,2,4]triazol-1-ylmethyl-benzoicacid methyl ester (CAB04121): 4-Benzyloxy-3-fluoro-benzenesulfonylchloride (301 mg, 1.0 mmol) was added to a solution of CAB04083 (232 mg,1.0 mmol) in pyridine (5 ml). The mixture was stirred for 5 hours atroom temperature, and then EtOAc (50 ml) and water (30 ml) were added.The organic layer was separated, washed with 2M KHSO₄ (2×30 ml) andbrine (20 ml), dried over Na₂SO₄ and concentrated under reducedpressure. The residue was crystallized from EtOAc/hexane to give finecolorless needles. Yield: 457 mg (92%).

¹H-NMR (400 MHz, DMSO-d₆) δ 3.83 (3H, s, —OCH₃), 5.24 (2H, s), 5.47 (2H,s), 7.29-7.64 (11H, m), 8.02 (1H, s), 8.67 (1H, s), 10.62 (1H, s);¹³C-NMR (100.5 MHz, DMSO-d₆) δ 52.2, 52.8, 69.9, 115.0, 120.7, 123.8,124.2, 127.0, 128.3, 128.5, 130.0, 130.7, 137.1, 138.0, 140.6, 144.9,152.5, 161.7, 165.5, 166.3; MS (AP+): m/z 497.3 (100%, [C₂₄H₂₂FN₄O₃S]⁺);HPLC (ACS80) t_(r)=1.893 min (97.8%).STX1515 (CAB04122)

3-(3-Fluoro-4-hydroxy-benzenesulfonylamino)-5-[1,2,4]triazol-1-ylmethyl-benzoicacid methyl ester (CAB04122, STX1515): Pd/C (50 mg, 5%) was added to asolution of CAB04121 (298 mg, 0.60 mmol) in MeOH (10 ml) and THF (10ml). The mixture was stirred under H₂-atmosphere at room temperature for18 hours. The catalyst filtered off and the solution was concentratedunder reduced pressure to give the title compound as white solid. Yield:241 mg (99%).

¹H-NMR (270 MHz, DMSO-d₆) δ 3.79 (3H, s, —OCH₃), 5.47 (2H, s), 6.95-7.05(1H, m), 7.27-7.58 (5H, m), 7.98 (1H, s), 8.63 (1H, s), 10.48 (1H, brs), 11.05 (1H, br s); MS (AP−): m/z 405.2 (100%, [C₁₇H₁₄FN₄O₅S]⁻); HRMS(FAB+) calcd for C₁₇H₁₆FN₄O₅S: 407.08255; found, 407.08227. HPLC (ACS80)t_(r)=1.793 min (92.1%).STX1528 (CAB04128)

3-(3-Fluoro-4-sulfamoyloxy-benzenesulfonylamino)-5-[1,2,4]triazol-1-ylmethyl-benzoicacid methyl ester (CAB04128, STX1528): Sulphamoyl chloride solution intoluene (3 ml, 0.7 M, 2.1 mmol) was concentrated under reduced pressure.The residue was dissolved In DMA (5 ml) at 0° C. and CAB04122 (120 mg,0.295 mmol) was added to the solution. Stirring was continued for 18hours, EtOAc (50 ml) and water 30 ml were added, the organic layer wasseparated, washed with water (3×30 ml) and brine (20 ml), dried (Na₂SO₄)and concentrated under reduced pressure. The residue was purified byflash chromatography on silica (chloroform/acetone 1:1) to give thetitle compound as a light yellow solid. Yield 86 mg (60%).

¹H-NMR (270 MHz, DMSO-d₆) δ 3.86 (3H, s, —OCH₃), 5.52 (2H, s), 7.20-7.25(1H, m), 7.58-7.69 (3H, m), 7.74-7.76 (1H, m), 7.80-7.83 (1H, m), 7.93(3H, br s), 8.40 (1H, s), 9.57 (1H, br s); MS (FAB+): m/z 485.9 (100%,[M+H]⁺); HRMS (FAB+) calcd for C₁₇H₁₇FN₅O₇S: 486.05535; found,486.05566. HPLC (ACS80) t_(r)=1.740 min (95.1%).Synthesis of STX1829

CAB04124

5-(4-Benzyloxy-benzenesulfonylamino)-isophthalic acid dimethyl ester(CAB04124): 4-Benzyloxy-benzenesulfonyl chloride (7.35 g, 26.0 mmol) wasadded in small portions to a suspension of 5-amino isophtalic aciddimethyl ester (5.23 g, 25.0 mmol) in DCM (100 ml) and pyridine (10 ml).The resulting clear yellow solution was stirred for 2 hours at roomtemperature, the volatile solvents were removed under reduced pressure,the solid residue was suspended in MeOH (125 ml) and the mixture washeated to reflux for 10 minutes. The off white solid was filtered off,washed with water and cold MeOH and dried under high vacuum. Yield 10.93g (96%).

¹H-NMR (400 MHz, DMSO-d₆) δ 3.87 (6H, s, 2×—OCH₃), 5.13 (2H, s), 7.17(2H, d, J=9.0 Hz), 10.77 (1H, br s); ¹³C-NMR (100.5 MHz, DMSO-d₆) δ53.1, 70.2, 115.8, 124.2, 125.0, 128.4, 128.6, 128.9, 129.3, 131.1,131.6, 136.6, 139.6, 162.3, 165.3; MS (FAB+): m/z 456.2 (30%,[C₂₃H₂₂NO₇S]⁺), 71.0 (100%); HRMS (FAB+) calcd for C₂₃H₂₁NO₇: 455.09940;found, 455.10091. HPLC (ACS80) t_(r)=2.059 min (99.8%).CAB04125

5-[(4-Benzyloxy-benzenesulfonyl)-methyl-amino]-isophthalic acid dimethylester (CAB04125): A mixture of CAB04124 (10.476 g, 23.0 mmol), K₂CO₃(6.90 g, 50 mmol), methyl iodide (3.55 g, 25 mmol) and DMF (100 ml) wasstirred vigorously for 16 hours. The reaction mixture was poured intocrushed ice; the white precipitate was collected, washed with water andmethanol and dried under high vacuum. Yield 10.58 g (98%).

¹H-NMR (400 MHz, DMSO-d₆) δ 3.20 (3H, s, NCH₃), 3.90 (6H, s, 2×OCH₃),5.20 (2H, s), 7.19 (2H, d, J=9.0 Hz), 7.34-7.50 (7H, m), 7.92 (2H, d,J=1.6 Hz), 837 (1H, t, J=1.6 Hz); ¹³C-NMR (100.5 MHz, DMSO-d₆) δ 37.8,53.2, 70.3, 115.8, 127.5, 128.1, 128.4, 128.6, 129.0, 130.2, 130.8,131.4, 136.6, 142.8, 162.7, 165.2; MS (FAB+): m/z 470.2 (100%, [M+H]⁺);HRMS (FAB+) calcd for C₂₄H₂₃NO₇S: 469.11952; found, 469.11952. HPLC(ACS80) t_(r)=2.350 min (99.9%).CAB04126

5-[(4-Benzyloxy-benzenesulfonyl)-methyl-amino]-isophthalic acidmonomethyl ester (CAB04126): 2 M NaOH (10 ml) was added to a solution ofCAB04125 (9.57 g, 20.0 mmol) in THF (75 ml) and MeOH (75 ml). Themixture was stirred for 48 hours at room temperature and poured into amixture of water (500 ml) and conc. HCl (100 ml) with intensivestirring. After a short period of time a white, crystalline preciptateformed, which was filtered off, washed with water and dried under highvacuum. The solid was recrystallised from EtOAc/hexane. Yield: 8.29 g(91%).

¹H-NMR (400 MHz, DMSO-d₆) δ 3.19 (3H, s, NCH₃), 3.89 (3H, s, —OCH₃),5.19 (2H, s), 7.20 (2H, d, J=8.9 Hz), 7.34-7.49 (7H, m), 7.86-7.88 (1H,m), 7.90-7.92 (1H, m), 8.37 (1H, m) (acid proton not resolved); ¹³C-NMR(100.5 MHz, DMSO-d₆) δ 37.9, 53.1, 53.2, 70.3, 115.8, 127.5, 128.4,128.6, 130.0, 130.2, 130.7, 130.9, 131.2, 132.7, 136.6, 142.6, 162.6,165.3, 166.2; MS (FAB+): m/z 456.1 (100%, [M+H]⁺); MS (APCI−): m/z 454.2(100%, [M−H]⁻); HRMS (FAB+) calcd for C₂₃H₂₁NO₇S: 455.10387; found,455.10266. HPLC (ACS80) t_(r)=2.285 min (>99%).CAB04129

3-[(4-Benzyloxy-benzenesulfonyl)-methyl-amino]-5-hydroxymethyl-benzoicacid methyl ester (CAB04129): Borane in THF (10 ml, 1M solution) wasadded slowly to a solution of CAB04126 (2.28 g, 5.0 mmol) in THF (50 ml)at 0° C. The mixture was stirred overnight, AcOH (5 ml) was addedcarefully to destroy the excess borane. The solvent was removed underreduced pressure, the residue was dissolved in EtOAc (EtOAc (50 ml) andwashed with water (30 ml) and conc. NaHCO₃-solution (2×30 ml), dried(Na₂SO₄), concentrated under reduced pressure and purified by flashchromatography on silica (EtOAc/hexane 1:1), R_(f): 0.31) to give awhite solid. Yield: 839 mg (38%).

¹H-NMR (270 MHz, CDCl₃) δ 1.90 (1H, t, J=5.9 Hz, —OH), 3.15 (3H, s,NCH₃), 3.88 (3H, s, OCH₃), 4.70 (2H, d, J=5.9 Hz), 5.09 (2H, s), 6.96(2H, d, J=8.9 Hz), 7.30-7.50 (8H, m), 7.68 (1H, s), 7.92 (1H, s); MS(FAB+): m/z 442.1 (100%, [M+H]⁺);CAB04130

3-[(4-Benzyloxy-benzenesulfonyl)-methyl-amino]-5-chloromethyl-benzoicacid methyl ester (CAB04130): A mixture of CAB04129 (442 mg, 1.0 mmol)and thionyl chloride was heated to reflux for 2 hours. The excessthionyl chloride was removed under reduced pressure, the residue waspurified by flash chromatography on silica (EtOAc/hexane 1: 3, R_(f):0.35) to give the title compound as a colorless oil, which wascrystallized from DCM/hexane. Yield: 308 mg (67%) fine colorlessneedles.

¹H-NMR (270 MHz, CDCl₃) δ 3.17 (3H, s, NCH₃), 3.89 (3H, s, OCH₃), 4.55(2H, s, CH₂Cl), 5.12 (2H, s), 6.99 (2H, d, J=8.6 Hz), 7.30-8.00 (10H,m); MS (APCI−): m/z 458.1 (100%, [M−H]⁻);CAB04132

3-[(4-Benzyloxy-benzenesulfonyl)-methyl-amino]-5-[1,2,4]triazol-1-ylmethyl-benzoicacid methyl ester (CAB04132): A mixture of CAB04130 (230 mg, 0.5 mmol),1,2,4-triazole (67 mg, 1.0 mmol), K₂CO₃ (690 mg, 5.0 mmol) and NaI (75mg, 0.5 mmol) an acetone (20 ml) was stirred vigorously for 16 hours.The mixture was diluted with EtOAc (100 ml) and water (50 ml), theorganic layer was separated, washed with brine (20 ml), dried (Na₂SO₄)and concentrated under reduced pressure. The residue was purified byflash chromatography (EtOAc, R_(f): 0.39) to give the title compound asa colorless oil. Yield: 222 mg (90%).

¹H-NMR (400 MHz, CDCl₃) δ 3.15 (3H, s, —NCH₃), 3.88 (3H, s, —OCH₃), 5.12(2H, s), 5.36 (2H, s), 6.98 (2H, d, J=9.0 Hz), 7.32-7.46 (9H, m),7.68-7.71 (1H, m), 7.84 (1H, m), 7.84 (1H, s), 7.97 (1H, s), 8.15 (1H,s); ¹³C-NMR (100.5 MHz, DMSO-d₆) δ 37.3, 52.1, 52.3, 70.0, 114.6, 126.3,127.1, 127.2, 127.3, 128.0, 128.4, 129.5, 130.4, 1313, 135.4, 135.7,142.4, 142.9, 152.0, 162.0, 165.2;STX1828 (CAB04133)

3-[(4-Hydroxy-benzenesulfonyl)-methyl-amino]-5-[1,2,4]triazol-1-ylmethyl-benzoicacid methyl ester (CAB04133, STX1828): Pd/C (50 mg, 5%) was added to asolution of CAB04132 (166 mg, 0.34 mmol) in MeOH (30 ml). The mixturewas stirred under H₂-atmosphere at room temperature for 24 hours. Thecatalyst filtered off and the solution was concentrated under reducedpressure to give the title compound as white solid. Yield: 136 mg(100%).

¹H-NMR (300 MHz, DMSO-d₆) δ 3.07 (3H, s, —NCH₃), 3.83 (3H, s, —OCH₃),5.47 (2H, s), 6.85 (2H, d, J=8.8 Hz), 7.28 (2H, d, J=8.8 Hz) 7.36-7.37(1H, m), 7.57-7.58 (1H, m), 7.76 (1H, s), 8.00 (1H, s), 8.63 (1H, s),10.58 (1H, br s, —OH); MS (APCI−): m/z 401.2 (100%, [M−H]⁻); HPLC(ACS90) t_(r)=2.698 min (92.8%).STX1829 (CAB04143)

3-[Methyl-(4-sulfamoyloxy-benzenesulfonyl)-amino]-5-[1,2,4]triazol-1-ylmethyl-benzoicacid methyl ester (CAB04143, STX1829): Sulphamoyl chloride solution intoluene (3 ml, 0.7 M, 2.1 mmol) was concentrated under reduced pressure.The residue was dissolved In DMA (5 ml) at 0° C. and CAB04133 (95 mg,0.236 mmol) was added to the solution. Stirring was continued for 18hours, EtOAc (50 ml) and water 30 ml were added, the organic layer wasseparated, washed with water (3×30 ml) and brine (20 ml), dried (Na2SO₄)and concentrated under reduced pressure. The residue was purified byflash chromatography on silica (chloroform/acetone 1:1) to give thetitle compound as a light as colorless foam. Yield 91 mg (80%).

¹H-NMR (400 MHz, CDCl₃) δ 3.15 (3H, s, NCH₃), 3.90 (3H, s, OCH₃), 5.34(2H, s, CH₂), 6.48 (1H, s), 7.20 (2H, brs, —NH₂), 7.32 (2H, d, J=8.9Hz), 7.41 (2H, d, J=8.9 Hz), 7.84-7.87 (3H, m), 7.99 (1H, s); ¹³C-NMR(100.5 MHz, CDCl₃) δ 38.3, 52.4, 52.7, 123.1, 127.8, 129.4, 129.5,131.9, 134.2, 136.6, 142.6, 143.5, 151.3, 153.5, 165.6. MS (APCI−): m/z480.2 (100%, [M−H]⁻); HPLC (ACS0) t_(r)=1.828 min (>99%).Synthesis of STX1830

STX1513 (CAB04120)

3-(4-Benzyloxy-benzoylamino)-5-[1,2,4]triazol-1-ylmethyl-benzoic acidmethyl ester (CAB04120, STX1513): 4-Benzyloxy-benzoyl chloride (247 mg,1.0 mmol) was added to a solution of CAB04038 (232 mg, 1.0 mmol) inpyridine (5 ml). The mixture was stirred for 5 hours at roomtemperature, and then EtOAc (50 ml) and water (30 ml) were added. Theorganic layer was separated, washed with 2M KHSO₄ (2×30 ml) and brine(20 ml), dried over Na₂SO₄ and concentrated under reduced pressure. Theresidue was crystallized from EtOAc/hexane to give a white powder.Yield: 425 mg (96%).

¹H-NMR (400 MHz, DMSO-d₆) δ 3.88 (3H, s, —OCH₃), 5.23 (2H, s), 5.53 (2H,s), 7.17 (2H, d, J=8.6 Hz), 7.34-7.51 (5H, m), 7.62-7.64 (1H, m), 7.98(2H, d, J=8.6 Hz), 8.00-8.02 (1H, m), 8.04 (1H, s), 8.41-8.42 (1H, m),8.74 (1H, s), 10.37 (1H, s, NH); ¹³C-NMR (100.5 MHz, DMSO-d₆) δ 52.2,52.8, 69.9, 115.0, 120.7, 123.8, 124.2, 127.0, 128.3, 128.5, 130.0,130.7, 137.1, 138.0, 140.6, 144.9, 152.5, 161.7, 165.5, 166.3; MS(FAB+): m/z 443.1 (100%, [C₂₅H₂₃N₄O₄]⁺); HRMS (FAB+) calcd forC₁₇H₁₆ClN₄O₅S: 443.17193; found, 443.17252. HPLC (ACS80) t_(r)=2.038 min(98.8%).STX1516 (CAB04123)

3-(4-Hydroxy-benzoylamino)-5-[1,2,4]triazol-1-ylmethyl-benzoic acidmethyl ester (CAB04123, STX1516): Pd/C (50 mg, 5%) was added to asolution of CAB04120 (265 mg, 0.60 mmol) in MeOH (10 ml) and THF (10ml). The mixture was stirred under H₂-atmosphere at room temperature for18 hours. The catalyst filtered off and the solution was concentratedunder reduced pressure to give the title compound as white solid. Yield:207 mg (98%).

¹H-NMR (270 MHz, DMSO-d₆) δ 3.85 (3H, s, —OCH₃), 5.50 (2H, s), 6.86 (2H,d, J=8.4 Hz), 7.59 (1H, s), 7.86 (2H, d, J=8.4 Hz), 7.97 (1H, s), 8.02(1H, s), 8.39 (1H, s), 8.71 (1H, s), 10.15 (1H, s), 10.24 (1H, s); MS(FAB+): m/z 353.2 (100%, [C₁₈H₁₇N₄O₄]⁺); MS (AP−): m/z 351.2 (100%,[C₁₈H₁₅N₄O₄]⁻); HPLC (ACS80) t_(r)=1.851 min (>99%).STX1830 (CAB04167)

3-(4-Sulfamoyloxy-benzoylamino)-5-[1,2,4]triazol-1-ylmethyl-benzoic acidmethyl ester (CAB04167, STX1830): Sulphamoyl chloride solution intoluene (3 ml, 0.7 M, 2.1 mmol) was concentrated under reduced pressure.The residue was dissolved In DMA (5 ml) at 0° C. and CAB04123 (80 mg,0.227 mmol) was added to the solution. Stirring was continued for 18hours, EtOAc (50 ml) and water 30 ml were added, the organic layer wasseparated, washed with water (3×30 ml) and brine (20 ml), dried (Na₂SO₄)and concentrated under reduced pressure. The residue was dissolved in asmall amount of EtOAc and precipitated by addition of Et₂O and hexane togive the title compound as a white solid. Yield 81 mg (82%).

¹H-NMR (270 MHz, DMSO-d₆) δ 3.87 (3H, s, —OCH₃), 5.53 (2H, s), 7.42 (2H,d, J=8.6 Hz), 7.65 (1H, s), 7.99 (1H, s), 8.03 (1H, s), 8.06 (2H, d,J=8.6 Hz), 8.19 (2H, s, —NH₂), 8.42 (1H, s), 8.72 (1H, s), 10.57 (1H, s,—NH); MS (APCI+): m/z 432.3 (100%, [M+H]⁺); HPLC (ACS90) t_(r)=1.739 min(99.7%).Synthesis of STX1522

4-Bromo-2-bromomethylbenzonitrile (TJA01043)

C₈H₅Br₂N MW 274.94

4-Bromo-2-methylbenzonitrile (5.00 g, 25.5 mmol), N-bromosuccinimide(4.99 g, 28.1 mmol), benzyl peroxide (0.198 g, 0.816 mmol) and carbontetrachloride (100 mL) were loaded to a r.b. flask and set to reflux(79° C.) for 6 h. Once cooled the succinimide was filtered off andcarbon tetrachloride removed via a dry ice-acetone cooled rotaryevaporator. The residues were dissolved in dichloromethane (100 mL) andwashed with distilled H₂O (50 mL×3) and brine (50 mL×2). Dried overNa₂SO₄ and solvent removed in vacuo to leave yellow residues. Columnchromatography (hexane/dichloromethane 60:40) eluted the title compoundas a yellow solid. Recrystallisation (cyclohexane) gave a whitecrystalline solid (5.07 g, 73%), mp 61.7-77.2° C. which was then usedwithout further purification; Rf 0.30 (hexane/dichloromethane 60:40),c.f. 0.36 (dibromobenzylbromide), 0.36 (4-bromo-2-methylbenzonitrile).

HPLC (60% CH₃CN in H₂O) R_(t)3.130 (50.62%), 2.701 (42.38%,dibromobenzylbromide); MS (EI), m/z 274.0 (M⁺, 34%).

4-Bromo-2-(1,2,4)triazol-1-ylmethyl-benzonitrile (TJA01046, STX1454)

C₁₀H₇BrN₄ MW 263.10

TJA01043 (5.00 g, 18.2 mmol), 1,2,4-triazole (1.89 g, 27.3 mmol),potassium carbonate (2.52 g, 18.2 mmol), potassium iodide (0.178 g, 1.07mmol) and acetone (150 mL) were loaded to an r.b. flask. With vigorousstirring this mixture was set to reflux (60° C.) for 4 h. The reactionmixture was allowed to cool and acetone was removed in vacuo. Theresidues were taken up in ethyl acetate (50 mL) and washed withdistilled water (50 mL×2), 1M NaOH (50 mL×1) and brine (50 mL×2). Driedover Na₂SO₄ and solvent removed in vacuo to leave orange/yellowresidues. Flash chromatography (50 g column, Flashmaster II, methodTJA01046) eluted dibromobenzylbromide and the title compound as a yellowsolid. Recrystallisation (ethyl acetate/hexane 1:6) gave a yellowcrystalline solid (1.58 g, 66%), mp 106.8-107.6° C.; R_(f)0.55 (ethylacetate).

¹H NMR (270 MHz, CDCl₃) δ 5.51 (2H, s, ArCH₂N), 7.50 (1H, s, ArH),7.53-7.56 (1H, d, J=8.8 Hz, ArH), 7.59-7.62 (1H, dd, J=1.7 & 8.5 Hz,ArH), 7.99 (1H, s, C₂H₂N₃) and 8.27 (1H, s, C₂H₂N₃); ¹³C NMR (100.5 MHz,CDCl₃) δ 50.7 (CH₂), 110.6, 116.3, 128.8, 132.8, 132.9, 134.2, 139.8,143.9 and 153.0; HPLC (80% CH₃CN in H₂O) t_(r)=1.947 min (100%); MS(EI), m/z 264.71 (⁸¹BrM⁺, 100%), 262.71. (⁷⁹BrM⁺, 99), 195.56 ((⁸¹BrM⁺−(C₂H₂N₃), 81), 193.56 ((⁷⁹BrM−(C₂H₂N₃)⁺, 80); Anal. Calc. for C₁₀H₇BrN₄: C 45.65, H 2.68, N 21.30. Found: C 45.60, H 2.70, N 20.9%.

4′-Hydroxy-3-[1,2,4]triazol-1-ylmethyl-biphenyl-4-carbonitrile(TJA01065, STX1520)

C₁₆H₁₂N₄O MW 276.30

A 10 mL microwave vial was loaded with TJA01046 (0.100 g, 0.380 mmol),4-hydroxyphenylboronic acid (0.079 g, 0.570 mmol), potassium carbonate(0.131 g, 0.950 mmol), tetrabutylammonium bromide (0.126 g, 0.380 mmol),Pd(OAc)₂ (0.001-0.002 g, 2-3 mol %), ethanol (1.5 mL) and distilledwater (3.5 mL). The vial was sealed and loaded (with no prior degassing)into a CEM Discover Microwave. After a run time of 3 min at 120° C.complete conversion was evident by tlc (ethyl acetate). The reactionmixture was allowed to cool and ethyl acetate (50 mL) added. This wasthen washed with distilled water (3×25 mL) and brine (2×25 mL). Theorganic layer was dried over Na₂SO₄, filtered and solvent removed invacuo to leave a yellow/brown residue. The crude product was purified byflash chromatography (20 g column, Flashmaster II, method insol3)eluting the title compound as a white solid (0.082 g, 79%), mp203.4-203.6° C. R_(f): 0.43 (ethyl acetate).

¹H NMR (270 MHz, DMSO-d₆) δ 5.62 (2H, s, ArCH₂N), 6.85-6.88 (2H, d,J=8.7 Hz, ArH), 7.51-7.55 (2H, d, J=8.7 Hz, ArH), 7.67-7.89 (3H, m,ArH), 7.99 (1H, s, NCHN), 8.71 (1H, s, NCHN) and 9.83 (1H, s, ArOH); ¹³CNMR (100.5 MHz, DMSO-d₆) δ 51.0, 109.2, 116.5, 117.8, 126.5, 127.5,128.8, 134.3, 139.9, 145.4, 152.6 and 159.0; HPLC (80% CH₃CN in H₂O)t_(r)=1.783 (97.91%); LCMS (APCI), m/z 275.22 (M⁺+H, 100%);

Sulfamic acid 4′-cyano-3′-[1,2,4]triazol-1-ylmethyl-biphenyl-4-yl ester(TJA01068, STX1522)

C₁₆H₁₃N₅O₃S MW 355.37

Sulfamoyl chloride in toluene (0.35 M, 5.17 mL) was transferred to anr.b. flask and the solvent removed under vacuum at 30° C. On cooling awhite solid formed to which was added N,N-dimethylacetamide (1.5 mL) toform a colourless solution. TJA01065 (0.100 g, 0.361 mmol) was added andthe solution left to stir at room temperature under N_(2 (g)) for 18 h.The reaction mixture was then poured into distilled H₂O (30 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL×2). Dried overNa₂SO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a white solid (0.106 g, 82%); mp 179.2-179.3° C. R_(f): 0.25(dichloromethane/acetone 80:20).

¹H NMR (270 MHz, DMSO-d₆) δ 5.67 (2H, s, ArCH₂N), 7.38-7.41 (2H, d,J=8.7 Hz, ArH), 7.76-7.79 (2H, d, J=8.7 Hz, ArH), 7.82-7.86 (2H, s,ArH), 7.96-7.99 (1H, d, J=8.2 Hz, ArH), 8.01 (1H, s, C₂H₂N₃), 8.08 (2H,s, ArOSO₂NH₂) and 8.72 (1H, s, C₂H₂N₃); ¹³C NMR (100.5 MHz, DMSO-d₆) δ50.9 (CH₂), 110.7, 117.5, 123.4, 127.6, 128.5, 129.1, 134.5, 136.7,140.2, 144.4, 145.4, 151.2 and 152.6; HPLC (80% CH₃CN in H₂O)t_(r)=1.725 (100%); LCMS (APCI), m/z 356.27 (M⁺+H, 100%), 277.24((M⁺+H,)—OSO₂NH₂, 29).Synthesis of STX1523

1-Bromo-3-bromomethyl-5-methylbenzene (TJA01023)

C₈H₈Br₂ MW 263.96

To a solution of sodium bromate (24.4 g, 162 mmol) in distilled H₂O (40mL) was added 5-bromo-m-xylene (10.0 g, 54.0 mmol) in cyclohexane (108mL). To this clear mixture a solution of sodium hydrogen sulphate (30.8g, 162 mmol) in distilled H₂O (81 mL) was added drop wise with vigorousstirring over 60 min. The reaction mixture was stirred for a further 3 hat room temperature. The ethyl acetate was separated and diethyl ether(100 mL) added. This was then washed with saturated Na2SO_(3(aq)) (1×100mL), distilled water (2×100 ml) and brine (2×100 mL). Dried over Na₂SO₄and solvent removed in vacuo to leave a clear syrup. Columnchromatography (hexane) eluted the title compound as a clear oil thatcrystallised on standing to give a white crystalline solid that was usedwithout further purification (8.45 g, 60%); R_(f) 0.52 (hexane), c.f0.52 (dibromobenzylbromide), 0.45 (1,5-dibenzylbromide), 0.6(5-bromo-m-xylene).

¹H NMR (270 MHz, CDCl₃) δ 2.31 (3H, s, ArCH₃), 4.38 (2H, s, ArCH₂Br),7.11 (1H, s, ArH), 7.25 (1H, s, ArH) and 7.32 (1H, s, ArH); HPLC (60%CH₃CN in H₂O) t_(r)=3.877 (67%), 4.644 (31%, dibromobenzylbromide).

(3-Bromo-5-methyl-phenyl)acetonitrile (TJA01029)

C₉H8BrN MW 210.07

TJA01023 (11.3 g, 42.7 mmol), potassium cyanide (3.34 g, 51.2 mmol) andtetrabutylammonium bromide (0.700 g, 2.10 mmol) were loaded to an r.b.flask together with dichloromethane (60 mL) and distilled water (15 mL).With vigorous stirring the reaction mixture was set to reflux (45° C.)for 24 h. On cooling the organic fraction was separated and washed withdistilled water (50 mL×2) and brine (50 mL×2) then dried over Na₂SO₄ andsolvent removed in vacuo to leave a red/orange oil. Columnchromatography initially eluting with hexane separated thedibromobenzylbromide impurity. Further elution withhexane/dichloromethane (50:50) gave the title compound as a clear yellowoil (6.63 g, 74%).

R_(f): 0.54 (hexane/dichloromethane 50:50) ¹H NMR (270 MHz, CDCl₃) δ2.31 (3H, s, ArCH₃), 3.66 (2H, s, ArCH₂CN), 7.06 (1H, s, ArH), 7.25 (1H,s, ArH) and 7.27 (1H, s, ArH); ¹³C NMR (100.5 MHz, CDCl₃) δ 21.1, 23.2,117.4, 122.8, 127.4, 128.1, 131.7, 131.9 and 141.1; HPLC (80% CH₃CN inH₂O) t_(r)=2.278 (72.5%); LCMS (APCI), m/z 211.78 (⁸¹BrM⁺, 53%), 209.78(⁷⁹BrM⁺, 55), 184.83 (⁸¹BrM⁺−CN, 80), 182.83 (⁷⁹BrM⁺−CN, 76).

2-(3-Bromo-5-methylphenyl)-2-methyl-propionitrile (TJA01035)

C₁₁H₁₂BrN MW 238.13

To a dry r.b. flask purged with N₂ (g) was added TJA01029 (6.00 g, 28.6mmol) and dry THF (20 mL). With stirring this was cooled via anice-water bath and NaH (1.71 g, 71.4 mmol) was added gradually and thenleft to stir at 0° C. under N_(2 (g)) for 15 min. Iodomethane (3.91 mL,62.8 mmol) was then added dropwise. The resulting suspension was left tostir at room temperature for 16 h. Propan-2-ol (5 mL) was carefullyadded to the reaction mixture followed by dichloromethane (50 mL) andwashed with distilled H₂O (50 mL×2) and brine (50 mL×2). Dried overNa₂SO₄ and solvent removed in vacuo to leave a red/orange oil. Columnchromatography (hexane/dichloromethane 50:50) eluted the title compoundas a light yellow oil (5.65 g, 83%); R_(f): 0.38 (hexane/dichloromethane50:50), c.f. 0.26 (3-Bromo-5-methyl-phenyl)acetonitrile; ¹H NMR (270MHz, CDCl₃) δ 1.68 (6H, s, ArC(CH₃)₂CN), 2.33 (3H, s, ArCH₃), 7.20 (1H,s, ArH), 7.26 (1H, s, ArH) and 7.34 (1H, s, ArH); ¹³C NMR (100.5 MHz,CDCl₃) δ 21.3 (CH₃), 29.0 (CH₃), 36.9 (C), 122.8, 124.1, 124.9, 125.2,131.6, 140.9 and 143.4; HPLC (80% CH₃CN in H₂O) t_(r)=2.600 (89.65%);LCMS (APCI), m/z 239.93 (⁸¹BrM⁺, 3%), 237.93 (⁷⁹BrM⁺, 4), 212.92(⁸¹BrM⁺−CN, 100), 210.92 (⁷⁹BrM⁺−CN, 96), 157.89 (M⁺−Br, 18).

2-(3-Bromo-5-bromomethyl-phenyl)-2-methylpropionitrile (TJA01036)

C₁₁H₁₁Br₂N MW 317.03

To a solution of sodium bromate (9.51 g, 63.0 mmol) in distilled H₂O (32mL) was added TJA01035 (5.00 g, 21.0 mmol) in cyclohexane (42 mL). Tothis clear mixture a solution of sodium hydrogen sulphate (7.56 g, 63.0mmol) in distilled H₂O (63 mL) was added drop wise with vigorousstirring over 1 h. The reaction mixture was stirred for a further 4 h atroom temperature. The cyclohexane was separated and diethyl ether (100mL) added. This was then washed with distilled water (50 ml×2) and brine(50 mL×2). Dried over Na₂SO₄ and solvent removed in vacuo to leaveviscous orange oil. Column chromatography (hexane/dichloromethane 50:50)eluted starting material and the title compound as a clear viscous oil(3.64 g, 54%), R_(f) 0.55 (hexane/dichloromethane 50:50), c.f. 0.38(2-(3-bromo-5-methylphenyl)-2-methyl-propionitrile); ¹H NMR (270 MHz,CDCl₃) δ 1.71 (6H, s, ArC(CH₃)₂CN), 4.41 (2H, s, ArCH₂Br), 7.40-7.41(1H, t, J=1.7, ArH) and 7.48-7.51 (2H, m, ArH); HPLC (80% CH₃CN in H₂O)t_(r)=2.508 (83.05%); LRMS (FAB+), m/z 319.1 (⁸¹BrM⁺, 100%), 317.1(⁷⁹BrM⁺, 100).

2-(3-Bromo-5-[1,2,4-triazole-1-yl-methylphenyl)-2-methylpropionitrile(TJA01037, STX1453)

C₁₃H₁₃BrN₄ MW 305.18

TJA01036 (3.20 g, 10.1 mmol), 1,2,4-triazole (1.05 g, 15.2 mmol),potassium carbonate (1.40 g, 10.1 mmol), potassium iodide (0.10 g, 0.600mmol) and acetone (150 mL) were loaded to an r.b. flask. With vigorousstirring this mixture was set to reflux (60° C.) for 24 h. The reactionmixture was allowed to cool and acetone was removed in vacuo. Theresidues were taken up in ethyl acetate (50 mL) and washed withdistilled water (50 mL×2), 1M NaOH (50 mL×1) and brine (50 mL×2). Driedover Na₂SO₄ and solvent removed in vacuo to leave a yellow oil. Columnchromatography (ethyl acetate) eluted the title compound as a clearviscous oil that crystallised on standing to give a colourlesscrystalline solid (1.97 g, 64%), mp 70.9-71.8° C.; R_(f) 0.24 (ethylacetate).

¹H NMR (270 MHz, CDCl₃) δ 1.68 (6H, s, ArC(CH₃)₂CN), 5.33 (2H, s,ArCH₂N), 7.40-7.41 (2H, t, J=1.7, ArH), 7.54-7.55 (1H, t, J=1.7, ArH),7.99 (1H, s, C₂H₂N₃) and 8.12 (1H, s, C₂H₂N₃); ¹³C NMR (100.5 MHz,CDCl₃) δ 29.0 (CH₃), 37.0 (C), 52.6 (CH₂), 123.5, 123.7, 128.6, 130.4,137.7, 143.4, 144.5 and 152.6 (one overlapping peak); HPLC (60% CH₃CN inH₂O increasing to 95% over 10 min) t_(r)=2.293 (98.87%); MS (EI), m/z307.09 (⁸¹BrM⁺, 100%), 305.09 (⁷⁹BrM⁺, 99), 238.01 ((⁸¹BrM−(C₂H₂N₃)⁺,22), 236.01 ((⁷⁹BrM−(C₂H₂N₃)⁺, 24).

2-(4′-Hydroxy-5-[1,2,4]triazol-1-ylmethyl-biphenyl-3-yl)-2-methyl-propionitrile(TJA01067, STX1521)

C₁₉H₁₈N₄O MW 318.37

A 10 mL microwave vial was loaded with TJA01037 (0.200 g, 0.656 mmol),4-hydroxyphenylboronic acid (0.136 g, 0.984 mmol), potassium carbonate(0.227 g, 1.64 mmol), tetrabutylammonium bromide (0.218 g, 0.656 mmol),Pd(OAc)₂ (0.004-0.005 g, 2-3 mol %), ethanol (1.5 mL) and distilledwater (3.5 mL). The vial was sealed and loaded (with no prior degassing)into a CEM Discover Microwave. After a run time of 3 min at 120° C.complete conversion was evident by tlc (ethyl acetate). The reactionmixture was allowed to cool and ethyl acetate (50 mL) added. This wasthen washed with distilled water (25×3 mL) and brine (25 mL). Theorganic layer was dried over Na₂SO₄, filtered and solvent removed invacuo to leave a yellow/brown residue. The crude product was purified byflash chromatography (20 g column, Flashmaster II, method insol3)eluting the title compound as a pale yellow (0.216 g, 89%), mp65.8-68.1° C. R_(f): 0.28 (ethyl acetate).

¹H NMR (270 MHz, DMSO-d₆) δ 1.71 (6H, s, ArC(CH₃)₂CN), 5.49 (2H, s,ArCH₂N), 6.84-6.87 (2H, d, J=8.7 Hz, ArH), 7.38 (1H, s, ArH), 7.42 (1H,s, ArH), 7.44-7.48 (2H, d, J=8.7 Hz, ArH), 7.59 (1H, s, ArH), 8.00 (1H,s, C₂H₂N₃), 8.72 (1H, s, C₂H₂N₃) and 9.64 (1H, s, ArOH); ¹³C NMR (100.5MHz, DMSO-d₆) δ 28.8 (CH₃), 37.3 (C), 52.5 (CH₂), 116.3, 122.9, 123.3,125.0, 125.5, 128.5, 130.5, 138.0, 141.8, 143.0, 144.9, 152.3 and 158.0;HPLC (80% CH₃CN in H₂O) t_(r)=1.787 (99.55%); LCMS (APCI), m/z 317.29(M⁺−H, 100%).

Sulfamic acid3′-(cyano-dimethyl-methyl)-5′-[1,2,4]triazol-1-ylmethyl-biphenyl-4-ylester (TJA01069, STX1523)

C₁₉H₁₉N₅O₃S MW 397.45

Sulfamoyl chloride in toluene (0.35 M, 4.49 mL) was transferred to anr.b. flask and the solvent removed under vacuum at 30° C. On cooling awhite solid formed to which was added N,N-dimethylacetamide (4.0 mL) toform a colourless solution. TJA01067 (0.100 g, 0.314 mmol) was added andthe solution left to stir at room temperature under N_(2 (g)) for 20 h.The reaction mixture was then poured into distilled H₂O (50 mL) andextracted with ethyl acetate (50 mL×2). The organic layers were combinedand washed with distilled H₂O (50 mL×4) and brine (25 mL). Dried overNa₂SO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a white waxy solid (0.111 g, 86%); mp 74.3-76.1° C. R_(f): 0.21(dichloromethane/acetone 75:25).

¹H NMR (270 MHz, DMSO-d₆) δ 1.72 (6H, s, ArC(CH₃)₂)CN), 5.52 (2H, s,ArCH₂N), 7.35-7.38 (2H, d, J=8.7 Hz, ArH), 7.49 (2H, s, ArH), 7.67 (1H,s, ArH), 7.69-7.72 (2H, d, J=8.7 Hz, ArH), 7.99 (1H, s, C₂H₂N₃), 8.04(2H, s, ArOSO₂NH₂) and 8.71 (1H, s, C₂H₂N₃); ¹³C NMR (100.5 MHz,DMSO-d₆) δ 28.7 (CH₃), 37.3 (C), 52.3 (CH₂), 123.3, 123.8, 124.6, 124.9,126.3, 128.8, 138.3, 140.9, 143.3, 144.9, 150.5 and 152.4 (oneoverlapping signal); HPLC (80% CH₃CN in H₂O) t_(r)=1.747 (98.58%); LCMS(APCI), m/z 398.22 (M⁺+H, 100%), 319.24 ((M⁺+H,)—OSO₂NH₂, 15).Synthesis of STX1729

3-Bromomethyl-5-methyl-benzoic acid methyl ester (TJA01072)

C₁₀H₁₁BrO₂ MW 243.10

To a solution of sodium bromate (13.80 g, 91.5 mmol) in distilled H₂O(45.8 mL) was added methyl 3,5-dimethylbenzoate (5.00 g, 30.5 mmol) inethyl acetate (15.3 mL). To this mixture a solution of sodium hydrogensulphate (10.99 g, 91.5 mmol) in distilled H₂O (91.5 mL) was added dropwise with vigorous stirring over 1 h. The reaction mixture was stirredfor a further 4 h at room temperature. The ethyl acetate was separatedand diethyl ether (50 mL) added. This was then washed with saturatedNa₂SO_(3(aq)) (1×50 mL), distilled water (50 ml×2), and brine (50 mL×2).Dried over Na₂SO₄ and solvent removed in vacuo to leave a clear syrup.The syrup was dissolved with gentle heating in hexane (50 mL) and theresulting clear solution allowed to cool and then stand at 0° C. for 30min. A white ppt (3,5-bis-bromomethyl-benzoic acid methyl ester) wasremoved via filtration. The filtrate was reduced to a clear syrup.Column chromatography (hexane/ethyl acetate 10:1) eluted the titlecompound as a clear oil (6.3 g, 85%—of which 14% is methyl3,5-dimethylbenzoate); R_(f) 0.45 (hexane), c.f 0.3(3,5-bis-bromomethyl-benzoic acid methyl ester), 0.56 (methyl3,5-dimethylbenzoate).

¹H NMR (270 MHz, CDCl₃) δ 2.37 (3H, s, ArCH₃, 3.89 (3H, s, ArCO₂CH₃),4.47 (2H, s, ArCH₂Br), 7.38 (1H, s, ArH), 7.77 (1H, s, ArH) and 7.84(1H, s, ArH); ¹³C NMR (100.5 MHz, CDCl₃) δ 21.2 (CH₃), 32.7 (CH₃), 52.3(CH₂), 127.3, 130.3, 130.7, 134.2, 138.1, 139.0 and 166.7 (C═O);

3-Cyanomethyl-5-methyl-benzoic acid methyl ester (TJA01076)

C₁₁H₁₁NO₂ MW 189.21

TJA01072 (19.5 g, 80.1 mmol), potassium cyanide (6.26 g, 96.1 mmol) andtetrabutylammonium bromide (1.33 g, 4.00 mmol) were loaded to an r.b.flask together with dichloromethane (100 mL) and distilled water (40mL). With vigorous stirring the reaction mixture was set to reflux (45°C.) for 24 h. On cooling the organic fraction was separated and washedwith distilled water (100 mL×2) and brine (100 mL) then dried over MgSO₄and solvent removed in vacuo to leave a red/orange oil. Columnchromatography initially eluting with hexane separated thedibromobenzylbromide impurity. Further elution with hexane/ethyl acetate(50:50) gave the title compound as a pale yellow solid (11.39 g, 75%),mp 56.2-57.8° C.; R_(f): 0.08 (ethyl acetate) c.f. 0.45 (TJA01072) ¹HNMR (270 MHz, CDCl₃) δ 2.34 (3H, s, ArCH₃), 3.79 (2H, s, ArCH₂CN), 3.89(3H, s, ArCO₂CH₃), 7.34 (1H, s, ArH), 7.76 (1H, s, ArH) and 7.79 (1H, s,ArH); ¹³C NMR (100.5 MHz, CDCl₃) δ 21.2 (CH₃), 23.4 (CH₃), 52.3 (CH₂),117.6, 126.3, 130.0, 130.2, 131.0, 133.1, 139.5 and 166.7 (C═O); HPLC(90% CH₃CN in H₂O) t_(r)=2.001 (100%); LCMS (APCI), m/z 187.97 (M⁺−H,100%).

3-(Cyano-dimethyl-methyl)-5-methyl-benzoic acid methyl ester (TJA01077)

C₁₃H₁₅NO₂ MW 217.11

To a dry r.b. flask purged with N_(2 (g)) was added TJA01076 (7.00 g,37.0 mmol) and dry THF (20 mL). With stirring this was cooled via anice-water bath and NaH (2.22 g, 92.5 mmol) was added gradually and thenleft to stir at 0° C. under N_(2 (g)) for 15 min. Iodomethane (11.6 mL,81.4 mmol) was then added dropwise. The resulting suspension was left tostir at room temperature for 16 h. Propan-2-ol (5 mL) was carefullyadded to the reaction mixture followed by dichloromethane (50 mL) andwashed with distilled H₂O (50 mL×2) and brine (50 mL ). Dried over MgSO₄and solvent removed in vacuo to leave a red/orange oil. Columnchromatography (hexane/ethyl acetate 90:10) eluted the title compound asa light yellow solid (6.29 g, 78%), mp 53.8-55.9° C.; R_(f): 0.26(hexane/ethyl acetate 90:10); ¹H NMR (270 MHz, CDCl₃) δ 1.73 (6H, s,ArC(CH₃)₂CN), 2.41 (3H, s, ArCH₃), 3.91 (3H, s, ArCO₂CH₃), 7.51 (1H, s,ArH), 7.79 (1H, s, ArH) and 7.87 (1H, s, ArH); ¹³C NMR (100.5 MHz,CDCl₃) δ 21.4, 29.1, 37.1, 52.3, 123.1, 124.3, 129.7, 130.7, 130.8,139.2, 141.8 and 166.8 (C═O); HPLC (90% CH₃CN in H₂O) t_(r)=4.293(100%); LCMS (APCI), m/z 217.99 (M⁺+H, 83%), 191.99 ((M⁺+H,)—CN, 100).

3-Bromomethyl-5-(cyano-dimethyl-methyl)-benzoic acid methyl ester(TJA01079)

C₁₃H₁₄BrNO₂ MW 296.17

To a solution of sodium bromate (9.40 g, 62.2 mmol) in distilled H₂O (31mL) was added TJA01077 (4.50 g, 20.8 mmol) in ethyl acetate (21 mL). Tothis clear mixture a solution of sodium hydrogen sulphate (7.47 g, 62.2mmol) in distilled H₂O (124 mL) was added drop wise with vigorousstirring over 1 h. The reaction mixture was stirred for a further 4 h atroom temperature. The ethyl acetate was separated and diethyl ether (100mL) added. This was then washed with distilled water (50 ml×2) and brine(50 mL). Dried over MgSO₄ and solvent removed in vacuo to leave thetitle compound as a light yellow brown syrup (9.3 g, 75%—of which 33%starting material); R_(f) 0.45 (hexane/ethyl acetate 85:15), c.f 0.52(TJA01077), used without further purification.

HPLC (80% CH₃CN in H₂O) t_(r)=3.367 (85%); LCMS (APCI), m/z 298.08(⁸¹BrM⁺+H, 15%), 296.08 (⁷⁹BrM⁺+), 15), 272.01 ((81 BrM⁺+H,)—CN, 100),270.01 ((⁷⁹BrM⁺+H,)—CN, 100).

3-(Cyano-dimethyl-methyl)-5-[1,2,4]triazol-1-ylmethyl-benzoic acidmethyl ester (TJA01080)

C₁₅H₁₆N₄O₂ MW 284.32

TJA01079 (9.30 g, 31.4 mmol), 1,2,4-triazole (3.25 g, 47.1 mmol),potassium carbonate (4.34 g, 31.4 mmol), potassium iodide (0.31 g, 1.85mmol) and acetone (200 mL) were loaded to an r.b. flask. With vigorousstirring this mixture was set to reflux (60° C.) for 22 h. The reactionmixture was allowed to cool and acetone was removed in vacuo. Theresidues were taken up in ethyl acetate (50 mL) and washed withdistilled water (50 mL×3) and brine (50 mL). Dried over MgSO₄ andsolvent removed in vacuo to leave a dark brown syrup. Columnchromatography (hexane/ethyl acetate 85:15 then ethyl acetate) elutedTJA01077 (1.25 g) and the title compound as a brown solid (3.70 g, 60%),mp 78.0-79.4° C.; R_(f) 0.18 (ethyl acetate).

¹H NMR (270 MHz, CDCl₃) δ 1.72 (6H, s, ArC(CH₃)₂CN), 3.90 (3H, s,ArCO₂CH₃), 5.40 (2H, s, ArCH₂Br), 7.60 (1H, s, ArH), 7.85 (1H, s, ArH),7.98 (1H, s, C₂H₂N₃), 8.07 (1H, s, ArH) and 8.14 (1H, s, C₂H₂N₃); ¹³CNMR (100.5 MHz, CDCl₃) δ 29.0, 37.2, 52.6, 52.9, 123.7, 126.4, 128.5,129.3, 131.9, 136.2, 143.1, 153.0 and 165.9 (C═O) (one overlappingpeak); HPLC (80% CH₃CN in H₂O) t_(r)=1.901 (99.60%); LCMS (APCI), m/z285.11 (M⁺+H, 100%), 215.98 ((M⁺+H,)—C₂H₂N₃, 45).

2-(3-Hydroxymethyl-5-[1,2,4]triazol-1-ylmethyl-phenyl)-2-methyl-propionitrile(TJA01097)

C₁₄H₁₆N₄O MW 256.13

A 100 mL r.b. flask was loaded with TJA01080 (0.500 g, 1.76 mmol) andpolyethylene glycol 400 (6.0 g). The mixture was heated to 80° C. withstirring until a solution had formed. Sodium borohydride (0.200 g, 5.28mmol) was added carefully resulting in evolution of gas. The reactionmixture was stirred vigorously at 80° C. for 16 h. An extremely viscousglue formed that gradually dissolved in dichloromethane (50 mL) withheating (40° C.). This solution was washed with 1M HCl_((aq)) (10 mL)and then carefully neutralised with sodium bicarbonate. Washed withdistilled water (50 mL×4) and brine (50 mL), separated and dried overMgSO₄. Solvent removed in vacuo to leave a viscous yellow oil. Flashchromatography (20 g column, Flashmaster II, method TJA01097) eluted thetitle compound as a white crystalline solid (0.351 g, 78%), mp113.5-115.8° C.; R_(f): 0.12 (ethyl acetate), c.f. 0.24 (TJA01090b) and0.24 (TJA01080); ¹H NMR (270 MHz, CDCl₃) δ 1.70 (6H, s, ArC(CH₃)₂CN),2.19-2.28 (1H, t, J=5.5 Hz, ArCH₂OH), 4.69-4.71 (2H, d, J=5.5 Hz,ArCH₂OH), 5.35 (2H, s, ArCH₂N), 7.17 (1H, s, ArH), 7.29 (1H, s, ArH),7.44 (1H, s, ArH), 7.95 (1H, s, C₂H₂N₃) and 8.09 (1H, s, C₂H₂N₃); ¹³CNMR (67.8 MHz, CDCl₃) δ 29.2 (CH₃), 37.2 (C), 53.4 (CH₂), 64.5 (CH₂),123.9 (CH), 124.3 (C), 125.7 (CH), 135.9 (C), 142.7 (C), 143.1 (C),143.2 (CH) and 152.4 (CH) (one overlapping peak); HPLC (70% CH₃CN inH₂O) t_(r)=2.253 (100%); LCMS (APCI), m/z 257.23 (M⁺+H, 100%), 188.12((M⁺+H,)—C₂H₂N₃, 88)

Cyanomethyl-trimethyl-phosphonium iodide (TJA01110)

C₅H₁₁INP MW 243.03

Trimethylphosphine in THF (1M, 20.0 mL, 20.0 mmol) at 0° C. underN_(2 (g)) was diluted with anhydrous toluene (40 mL). Iodoacetonitrile(1.40 mL, 19.4 mmol) was added dropwise with vigorous stirring forming awhite ppt. The mixture was allowed to warm to r.t. and left to stir for40 h. The mixture was filtered and washed with toluene to give a whitesolid which was dried under vacuum. Recrystallisation (acetonitrile)provided the title compound as a white crystalline solid (3.23 g, 66%),¹H NMR (300 MHz, DMSO-d₆) δ 2.01-2.06 (9H, d, J=15.3 Hz, P(CH₃)₃),4.01-4.07 (2H, d, J=16.4 Hz, PCH₂CN); ³¹P NMR (121.5 MHz, DMSO-d₆) δ32.9.

Methanesulfonic acid2-chloro-4-[3-(cyano-dimethyl-methyl)-5-[1,2,4]triazol-1-ylmethyl-benzylsulfanyl]-phenylester (TJA01121)

C₂₁H₂₁ClN₄O₃S₂ MW 477.00

TJA01110 (0.114 g, 0.470 mmol) was added to a mixture of TJA01097 (0.100g, 0.390 mmol), methanesulfonic acid-2-chloro-4-mercapto-phenyl ester(0.136 g, 0.570 mmol), diisopropylethylamine (88.0 μL, 0.510 mmol) andpropionitrile (1.0 mL) in a dry 5 mL r.b. flask purged with N_(2 (g)).The mixture was then set to stir at 92° C. After 3 h a further 0.5equivalents of TJA01110 (0.047 g, 0.195 mmol), diisopropylethylamine(33.9 μL, 0.195 mmol) and methanesulfonicacid-2-chloro-4-mercapto-phenyl ester (0.047 g, 0.195 mmol) was added.This was repeated after 5 h. After 17 h the reaction was allowed tocool. Dichloromethane (20 mL) and distilled water (20 mL) were added andthe aqueous layer separated and extracted with dichloromethane (20mL×2). The organic fractions were combined and washed with brine (20mL), dried over MgSO₄ and solvent removed in vacuo to leave yellowresidues. Flash chromatography (20 g column, Flashmaster II, methodEtOAc) eluted the title compound as a yellow viscous oil (0.114 g, 61%),R_(f): 0.27.

¹H NMR (300 MHz, CDCl₃) δ 1.60 (6H, s, ArC(CH₃)₂CN), 3.19 (3H, s,ArOSO₂CH₃), 4.01 (2H, s, ArCH₂S), 5.26 (2H, s, ArCH₂N), 6.96 (1H, s,ArH), 7.07-7.12 (1H, dd, J=2.3 & 6.3 Hz, ArH), 7.19-7.26 (4H, m, ArH),7.92 (1H, s, C₂H₂N₃) and 8.04(1H, s, C₂H₂N₃); ¹³C NMR (100.5 MHz, CDCl₃)δ 29.1 (CH₃), 37.2 (CH₂), 38.9 (CH₃), 39.0 (CH₂), 53.1 (CH₂), 123.8(CH), 124.0 (C), 124.9 (CH), 126.0 (CH), 127.7 (CH), 130.0 (CH), 132.1(CH), 136.2 (C), 136.3 (C), 138.8 (C), 143.0 (C), 143.4 (CH) and 152.6(CH) (two overlapping signals); HPLC (90% CH₃CN in H₂O) t_(r)=1.991(100%); LCMS (APCI), m/z 479.26 (³⁷ClM⁺+H, 45%), 477.24 (³⁵ClM⁺+H, 100).

2-[3-(3-Chloro-4-hydroxy-phenylsulfanylmethyl)-5-[1,2,4]triazol-1-ylmethyl-phenyl]-2-methyl-propionitrile(TJA01123)

C₂₀H₁₉ClN₄OS MW 398.91

TJA01121 (0.100 g, 0.210 mmol) was dissolved in THF (2.5 mL) andmethanol (1.5 mL) to which 2M NaOH_((aq)) (0.52 mL) was added. Themixture was set to stir at room temp. for 1 h. THF was removed underreduced pressure and the residues taken up in ethyl acetate (20 mL) andwashed with 2M KHSO_(4 (aq)) (20 mL), distilled water (20 mL×2) andbrine (20 mL). The organic layer was then dried over MgSO₄ and solventremoved under reduced pressure to leave a colourless viscous oil. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a colourless viscous oil (0.071 g, 84%) that crystallised on standingto a white crystalline solid, mp 130.6-130.8° C.; R_(f): 0.31(dichloromethane/acetone 80:20); ¹H NMR (400 MHz, DMSO-d₆) δ 1.59 (6H,s, ArC(CH₃)₂CN), 4.10 (2H, s, ArCH₂SAr), 5.43 (2H, s, ArCH₂N), 6.85-6.87(1H, d, J=8.6 Hz, ArH), 7.07-7.10 (1H, dd, J=2.3 & 8.2 Hz, ArH), 7.14(1H, s, ArH), 7.20 (1H, s, ArH), 7.26 (1H, s, ArH), 7.34 (1H, s, ArH),8.00 (1H, s, C₂H₂N₃), 8.66 (1H, s, C₂H₂N₃) and 10.37 (1H, s, ArOH); HPLC(90% CH₃CN in H₂O) t_(r)=2.199 (85.27%); LCMS (APCI), m/z 401.38(³⁷ClM⁺+H, 30%), 399.43 (³⁵ClM⁺+H, 100).

Sulfinamic acid2-chloro-4-[3-(cyano-dimethyl-methyl)-5-[1,2,4]triazol-1-ylmethyl-benzylsulfanyl]-phenylester (TJA01124, STX1729)

C₂₀H₂₀ClN₅O₃S₂ MW 477.99

Sulfamoyl chloride in toluene (0.60 M, 1.05 mL) was transferred to anr.b. flask and the solvent removed under vacuum at 30° C. On cooling awhite solid formed to which was added N,N-dimethylacetamide (1.5 mL) toform a colourless solution. TJA01123 (0.050 g, 0.125 mmol) was added andthe solution left to stir at room temperature under N_(2 (g)) for 20 h.The reaction mixture was then poured into distilled H₂O (30 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL×2). Dried overNa₂SO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a colourless viscous oil (0.051 g, 85%), R_(f): 0.55(dichloromethane/acetone 80:20), c.f. 0.33 (TJA01123); ¹H NMR (270 MHz,DMSO-d₆) δ 1.62 (6H, s, ArC(CH₃)₂CN), 4.32 (2H, s, ArCH₂S), 5.44 (2H, s,ArCH₂N), 7.21 (1H, s, ArH), 7.34-7.39 (3H, m, ArH), 7.45 (1H, s, ArH),7.53-7.54 (1H, d, J=1.5 Hz, ArH), 7.99 (1H, s, C₂H₂N₃), 8.27 (2H, s,ArOSO₂NH₂) and 8.66 (1H, s, C₂H₂N₃); ¹³C NMR (100.5 MHz, DMSO-d₆) δ28.3, 36.2, 36.5, 51.8, 123.7, 124.1, 124.3, 125.5, 127.0, 127.5, 128.3,129.6, 135.5, 137.4, 138.5, 142.0, 144.2, 144.4 and 151.9; HPLC (90%CH₃CN in H₂O) t_(r)=1.929 (99.51%); LCMS (APCI), m/z 478.25 (³⁷ClM⁺−H,18%), 476.24 (³⁵ClM⁺−H, 40), 399.35 ((³⁷ClM⁺−H)—C₂H₂N₂, 40), 397.35((³⁵ClM⁺−H)—C₂H₂N₂, 100).Synthesis of STX1731

3-Bromomethyl-5-methyl-benzoic acid methyl ester (TJA01087)

C₁₀H₁₁BrO₂ MW 243.10

To a solution of sodium bromate (13.80 g, 91.5 mmol) in distilled H₂O(45.8 mL) was added methyl 3,5-dimethylbenzoate (5.00 g, 30.5 mmol) inethyl acetate (15.3 mL). To this mixture a solution of sodium hydrogensulphate (10.99 g, 91.5 mmol) in distilled H₂O (91.5 mL) was added dropwise with vigorous stirring over 1 h. The reaction mixture was stirredfor a further 4 h at room temperature. The ethyl acetate was separatedand diethyl ether (50 mL) added. This was then washed with saturatedNa₂SO_(3(aq)) (1×50 mL), distilled water (50 ml×2), and brine (50 mL×2).Dried over Na₂SO₄ and solvent removed in vacuo to leave a clear syrup.The syrup was dissolved with gentle heating in hexane (50 mL) and theresulting clear solution allowed to cool and then stand at 0° C. for 30min. A white ppt (3,5-bis-bromomethyl-benzoic acid methyl ester) wasremoved via filtration. The filtrate was reduced to a clear syrup.Column chromatography (hexane/ethyl acetate 10:1) eluted the titlecompound as a clear oil (6.3 g, 85%—of which 14% is methyl3,5-dimethylbenzoate); R_(f) 0.45 (hexane), c.f 0.3(3,5-bis-bromomethyl-benzoic acid methyl ester), 0.56 (methyl3,5-dimethylbenzoate).

¹H NMR (270 MHz, CDCl₃) δ 2.37 (3H, s, ArCH₃, 3.89 (3H, s, ArCO₂CH₃),4.47 (2H, s, ArCH₂Br), 7.38 (1H, s, ArH), 7.77 (1H, s, ArH) and 7.84(1H, s, ArH); ¹³C NMR (100.5 MHz, CDCl₃) δ 21.2 (CH₃), 32.7 (CH₃), 52.3(CH₂), 127.3, 130.3, 130.7, 134.2, 138.1, 139.0 and 166.7 (C═O);

3-Cyanomethyl-5-methyl-benzoic acid methyl ester (TJA01076)

C₁₁H₁₁NO₂ MW 189.21

TJA01087 (19.5 g, 80.1 mmol), potassium cyanide (6.26 g, 96.1 mmol) andtetrabutylammonium bromide (1.33 g, 4.00 mmol) were loaded to an r.b.flask together with dichloromethane (100 mL) and distilled water (40mL). With vigorous stirring the reaction mixture was set to reflux (45°C.) for 24 h. On cooling the organic fraction was separated and washedwith distilled water (100 mL×2) and brine (100 mL) then dried over MgSO₄and solvent removed in vacuo to leave a red/orange oil. Columnchromatography initially eluting with hexane separated thedibromobenzylbromide impurity. Further elution with hexane/ethyl acetate(50:50) gave the title compound as a pale yellow solid (11.39 g, 75%),mp 56.2-57.8° C.; R_(f): 0.08 (ethyl acetate) c.f. 0.45 (TJA01072) ¹HNMR (270 MHz, CDCl₃) δ 2.34 (3H, s, ArCH₃), 3.79 (2H, s, ArCH₂CN), 3.89(3H, s, ArCO₂CH₃), 7.34 (1H, s, ArH), 7.76 (1H, s, ArH) and 7.79 (1H, s,ArH); ¹³C NMR (100.5 MHz, CDCl₃) δ 21.2 (CH₃), 23.4 (CH₃), 52.3 (CH₂),117.6, 126.3, 130.0, 130.2, 131.0, 133.1, 139.5 and 166.7 (C═O); HPLC(90% CH₃CN in H₂O) t_(r)=2.001 (100%); LCMS (APCI), m/z 187.97 (M⁺−H,100%).

3-(1-Cyano-cyclobutyl)-5-methyl-benzoic acid methyl ester (TJA01098)

C₁₄H₁₅NO₂ MW 229.28

TJA01076 (1.50 g, 7.93 mmol) was loaded to a dry 25 mL r.b. flask whichwas subsequently purged with N_(2 (g)). To this was added dry DMF (10mL) and the solution cooled with stirring to 0° C. Sodium hydride (0.476g, 19.8 mmol) was carefully added resulting in a deep red coloration andevolution of gas. After 15 min at 0° C. 1,3-dibromopropane (0.960 mL,9.48 mmol) was added dropwise over 5 min. The reaction was allowed towarm to room temp. and left to stir for 1 h. Ethyl acetate (50 mL) wasadded to the reaction mixture and this was washed with distilled water(50 mL×4) and brine (50 mL). The organic layer was separated and driedover MgSO₄, filtered and solvent removed in vacuo. Column chromatography(hexane/EtOAc 70:30) eluted the title compound as a colourless oil(0.783 g, 43%), R_(f): 0.67 (hexane/EtOAc 70:30) ¹H NMR (270 MHz, CDCl₃)δ 2.00-2.23 (1H, m, CH₂), 2.35-2.52 (4H, m, ArCH₃ & CH₂), 2.55-2.69 (2H,m, CH₂), 2.77-2.89 (2H, m, CH₂), 3.91 (3H, s, ArCO₂CH₃), 7.39 (1H, s,ArH), 7.79 (1H, s, ArH) and 7.85 (1H, s, ArH); ¹³C NMR (100.5 MHz,CDCl₃) δ 17.1, 21.3, 34.6, 40.0, 52.3, 126.8, 124.2, 129.8, 130.8,131.1, 139.2, 140.2 and 166.7 (C═O); HPLC (90% CH₃CN in H₂O) t_(r)=2.578(99.99%); LCMS (APCI), m/z 230.19 (M⁺−H, 22%), 203.15 (M⁺−CN, 100).

3-Bromomethyl-5-(1-cyano-cyclobutyl)-benzoic acid methyl ester(TJA01102)

C₁₄H₁₄BrNO₂ MW 308.17

To a solution of sodium bromate (3.09 g, 20.5 mmol) in distilled H₂O (10mL) was added TJA01098 (0.783 g, 3.42 mmol) in ethyl acetate (7 mL). Tothis clear mixture a solution of sodium hydrogen sulphate (2.46 g, 20.5mmol) in distilled H₂O (20 mL) was added drop wise with vigorousstirring over 15 min. The reaction mixture was stirred for a further 4 hat room temperature. The ethyl acetate was separated and diethyl ether(100 mL) added. This was then washed with distilled water (25 ml×2) andbrine (25 mL). Dried over MgSO₄ and solvent removed in vacuo. Columnchromatography (hexane/ethyl acetate 75:25) eluted the title compound asa white crystalline solid (1.00 g, 96%), mp 93.9-94.6° C.; R_(f): 0.57(hexane/ethyl acetate 75:25), c.f 0.74 (TJA01084); ¹H NMR (270 MHz,CDCl₃) δ 2.04-2.21 (1H, m, CH₂), 2.31-2.54 (1H, m, CH₂), 2.57-2.70 (2H,dd, J=2.0 & 9.7 Hz, CH₂), 2.81-2.90 (2H, m, CH₂), 3.93 (3H, s,ArCO₂CH₃), 4.50 (2H, s, ArCH₂Br), 7.61 (1H, s, ArH) and 8.00 (2H, s,ArH); ¹³C NMR (100.5 MHz, CDCl₃) δ 17.1, 31.9, 34.7, 39.9, 52.5, 123.7,126.7, 129.7, 130.7, 131.6, 139.2, 141.1 and 166.0 (C═O); HPLC (80%CH₃CN in H₂O) t_(r)=3.486 (100%); LRMS (EI⁺), m/z 309.0 (⁸¹BrM⁺+H, 19%),307.0 (⁷⁹BrM⁺+H, 21%), 280.0 ((⁸¹BrM⁺+H,)—CN, 53), 278.0((⁷⁹BrM⁺+H,)—CN, 54), 200.0 (((M⁺+H,)—CN)—Br, 100).

3-(1-Cyano-cyclobutyl)-5-[1,2,4]triazol-1-ylmethyl-benzoic acid methylester (TJA01103)

C₁₆H₁₆N₄O₂ MW 296.13

TJA01102 (1.00 g, 3.24 mmol), 1,2,4-triazole (0.336 g, 0.191 mmol),potassium carbonate (0.448 g, 3.24 mmol), potassium iodide (0.032 g,0.191 mmol) and acetone (50 mL) were loaded to an r.b. flask. Withvigorous stirring this mixture was set to reflux (60° C.) for 16 h. Thereaction mixture was allowed to cool and acetone was removed in vacuo.The residues were taken up in ethyl acetate (50 mL) and washed withdistilled water (50 mL×3) and brine (50 mL). Dried over MgSO₄ andsolvent removed in vacuo to leave a dark brown syrup. Columnchromatography (hexane/ethyl acetate 50:50 then ethyl acetate) elutedthe title compound as a colourless viscous oil (0.410 g, 43%) R_(f):0.26 (ethyl acetate); ¹H NMR (270 MHz, CDCl₃) δ 2.04-2.21 (1H, m, CH₂),2.31-2.54 (1H, m, CH₂), 2.57-2.70 (2H, dd, J=2.0 & 9.7 Hz, CH₂),2.81-2.90 (2H, m, CH₂), 3.93 (3H, s, ArCO₂CH₃), 4.50 (2H, s, ArCH₂Br),7.61 (1H, s, ArH) and 8.00 (2H, s, ArH); ¹³C NMR (100.5 MHz, CDCl₃) δ17.1, 31.9, 34.7, 39.9, 52.5, 123.7, 126.7, 129.7, 130.7, 131.6, 139.2,141.1 and 166.0 (C═O); HPLC (85% CH₃CN in H₂O) t_(r)=2.089 (99.47%);LCMS (APCI), m/z 297.48 (M⁺+H, 100%), 214.33 ((M⁺+H)—CH₂C₂H₂N₃, 50).

1-(3-Hydroxymethyl-5-[1,2,4]triazol-1-ylmethyl-phenyl)-cyclobutanecarbonitrile(TJA01104)

C₁₅H₁₆N₄O MW 268.31

A 100 mL r.b. flask was loaded with TJA01103 (0.410 g, 1.38 mmol) andpolyethylene glycol 400 (5.0 g). The mixture was heated to 80° C. withstirring until a solution had formed. Sodium borahydride (0.157 g, 4.15mmol) was added carefully resulting in evolution of gas. The reactionmixture was stirred vigorously at 80° C. for 16 h. An extremely viscousglue formed that gradually dissolved in dichloromethane (50 mL) withheating (40° C.) This solution was washed with 1M HCl_((aq)) (10 mL) andthen carefully neutralised with sodium bicarbonate. Washed withdistilled water (50 mL×4) and brine (50 mL), separated and dried overMgSO₄. Solvent removed in vacuo to leave a colourless viscous oil. Flashchromatography (20 g column, Flashmaster II, method TJA01097) eluted thetitle compound as a colourless viscous oil (0.238 g, 64%), R_(f): 0.14(ethyl acetate), c.f. 0.28 (TJA01103); ¹H NMR (300 MHz, CDCl₃) δ2.08-2.15 (1H, m, CH₂), 2.20-2.23 (1H, t, J=5.8 Hz, ArCH₂OH), 2.46-2.55(1H, m, CH₂), 2.61-2.68 (2H, m, CH₂), 2.84-2.89 (2H, m, CH₂), 4.76-4.77(2H, d, J=5.8 Hz, ArCH₂OH), 5.41 (2H, s, ArCH₂N), 7.25 (1H, s, ArH),7.28 (1H, s, ArH), 7.45 (1H, s, ArH), 8.02 (1H, s, C₂H₂N₃), and 8.15(1H, s, C₂H₂N₃); ¹³C NMR (69.5 MHz, CDCl₃) δ 17.2 (C), 34.6 (CH₂), 40.1(CH₂), 53.3 (CH₂), 64.4 (CH₂), 124.3 (CH), 125.8 (CH), 135.9 (C), 141.1(C), 143.3 (CH) and 152.3 (CH) (two overlapping signals); HPLC (90%CH₃CN in H₂O) t_(r)=1.913 (100%); LCMS (APCI), m/z 269.45 (M⁺+H, 100%).

4-Methanesulfonyloxy-benzenesulfonic acid sodium salt (TJA01125)

C₇H₇NaO₆S₂ MW 274.13

SO₃ ⁻Na⁺

4-Hydroxybenzenesulfonic acid sodium salt dihydrate (11.6 g, 50.0 mmol)and sodium hydroxide (2.00 g, 50.0 mmol) were dissolved in distilledwater (50 mL) and the solution cooled to 0° C. Methane sulfonyl chloride(4.25 mL, 55.0 mmol) was added dropwise with stirring and the mixturethen allowed to warm to room temp. and left for 2 h. Brine (20 mL) wasadded and the solution left to stand for 1 h with the formation of whitecrystalline solid. The solids were filtered, recrystallised (brine), anddried under vacuum to give the title compound as a white crystallinesolid (7.81 g, 57%), mp>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 3.37 (3H,s, ArOSO₂CH₃) and 7.27-7.69 (4H, dd, J=8.7 & 17.5 Hz, AA′BB′); ¹³C NMR(69.5 MHz, DMSO-d₆) δ 37.9 (CH₃), 122.2 (CH), 128.0 (CH), 147.7 (C) and149.5 (C).

Methanesulfonic acid 4-chlorosulfonyl-phenyl ester (TJA01126)

C₇H₇ClO₅S₂ MW 270.71

Thionyl chloride (30 mL) was cooled to 0° C. Cautiously, with stirring,TJA01125 (7.80 g, 28.0 mmol) was added followed by DMF (0.5 mL). Thereaction mixture was subsequently heated to reflux (79° C.) for 1 h (oruntil evolution of gas has ceased) and then cooled. Thionyl chloride wasremoved in vacuo and the resulting yellow residues were taken up indichloromethane (50 mL) and distilled water (50 mL) carefully added. Theorganic layer was separated and washed with distilled water (50 mL×2)and brine (50 mL), dried over MgSO₄ and solvent removed in vacuo toleave yellow residues. Recystallisation (dichloromethane/hexane) gavethe title compound as a white crystalline solid (6.17 g, 80%), ¹H NMR(300 MHz, CDCl₃) δ 3.21 (3H, s, ArOSO₂CH₃) and 7.47-8.08 (4H, dd, J=8.8& 34.1 Hz, AA′BB′); ¹³C NMR (69.5 MHz, CDCl₃) δ 38.5 (CH₃), 123.3 (CH),129.6 (CH), 142.6 (C) and 153.6 (C); HPLC (90% CH₃CN in H₂O) t_(r)=2.489(99.63%).

Methanesulfonic acid 4-mercapto-phenyl ester (TJA01130)

C₇H₈O₃S₂ MW 204.27

A 50 mL r.b. flask was loaded with red phosphorus powder (0.715 g, 23.1mmol), iodine (0.039 g, 0.154 mmol) and acetic acid (7 mL). CautiouslyTJA01126 (2.50 g, 9.23 mmol) was added and the reaction mixture then setto reflux (118° C.) for 2 h. Distilled water (1.5 mL) was added and themixture left to reflux for a further 1 h. Reaction allowed to cool.Chloroform (30 mL) and distilled water (30 mL) were added. The organiclayer was separated and washed with distilled water (30 mL×3) and brine(30 mL). Dried over MgSO₄, filtered and solvent removed in vacuo toprovide the title compound as a white crystalline solid (1.74 g, 92%),R_(f): 0.79 (ethyl acetate); ¹H NMR (270 MHz, CDCl₃) δ 3.10 (3H, s,ArOSO₂CH₃), 3.51 (1H, s, ArSH) and 7.13-7.30 (4H, dd, J=8.9 & 39.8 Hz,AA′BB′); ¹³C NMR (69.5 MHz, CDCl₃) δ 37.4 (CH₃), 122.9 (CH), 130.7 (C),130.9 (CH), and 147.3 (C); HPLC (70% CH₃CN in H₂O) t_(r)=2.152 (96.26%);LCMS (APCI), nm/z 203.11 (M⁺−H, 30%), 124.01 (M⁺−SO₂CH₃, 100).

Methanesulfonic acid4-[3-(1-cyano-cyclobutyl)-5-[1,2,4]triazol-1-ylmethyl-benzylsulfanyl]-phenylester (TJA01145)

C₂₂H₂₂N₄O₃S₂ MW 454.57

A dry 5 mL r.b. flask purged with N_(2 (g)) was loaded with TJA01110(0.107 g, 0.447 mmol), TJA01104 (0.100 g, 0.373 mmol), TJA01130 (0.114,0.539 mmol), diisopropylethylamine (84.5 μL, 0.485 mmol) andpropionitrile (1.0 mL). The mixture was then set to stir at 93° C. After2 h a further 1 equivalent of TJA01110 (0.091 g, 0.373 mmol) anddiisopropylethylamine (65.0 μL, 0.373 mmol) were added. After 5 h thereaction was allowed to cool. Dichloromethane (20 mL) and distilledwater (20 mL) were added and the aqueous layer separated and extractedwith dichloromethane (20 mL×2). The organic fractions were combined andwashed with brine (20 mL), dried over MgSO₄ and solvent removed in vacuoto leave yellow residues. Column chromatography (ethyl acetate) elutedthe title compound as a yellow viscous oil (0.151 g, 92%), R_(f): 0.35(ethyl acetate); ¹H NMR (270 MHz, CDCl₃) δ 1.99-2.03 (1H, m, CH₂),2.32-2.58 (3H, m, CH₂), 2.71-2.85 (2H, m, CH₂), 3.15 (3H, s, ArOSO₂CH₃),4.06 (2H, s, ArCH₂S), 5.29 (2H, s, ArCH₂N), 6.97 (1H, s, ArH), 7.08-7.35(6H, m, ArH), 7.97 (1H, s, C₂H₂N₃) and 8.08 (1H, s, C₂H₂N₃); ¹³C NMR(69.5 MHz, CDCl₃) δ 17.1 (C), 34.5 (CH₂), 37.7 (CH₃), 39.2 (CH₂), 39.8(CH₂), 53.1 (CH₂), 122.7 (CH), 124.0 (C), 124.2 (CH), 126.4 (CH), 127.7(CH), 132.5 (CH), 134.6 (C), 136.1 (C), 139.3 (C), 141.2 (C), 143.4(CH), 148.0 (C) and 152.4 (CH); HPLC (90% CH₃CN in H₂O) t_(r)=1.859(100%); LCMS (APCI), m/z 455.40 (M⁺+H, 100%).

1-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-((4-hydroxyphenylthio)methyl)phenyl)cyclobutanecarbonitrile(TJA01151)

C₂₁H₂₀N₄OS MW 376.47

TJA01145 (0.133 g, 0.293 mmol) was dissolved in THF (4 mL) and methanol(2 mL) to which 2M NaOH_((aq)) (0.73 mL) was added. The mixture was setto stir at room temp. for 12 h. THF was removed under reduced pressureand the residues taken up in ethyl acetate (20 mL) and washed with 2MKHSO_(4 (aq)) (20 mL), distilled water (20 mL×2) and brine (20 mL). Theorganic layer was then dried over MgSO₄ and solvent removed underreduced pressure to leave a colourless viscous oil. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a colourless viscous oil (0.091 g, 82%), R_(f): 0.25(dichloromethane/acetone 80:20); ¹H NMR (270 MHz, CDCl₃) δ 1.93-2.03(1H, m, CH₂), 2.31-2.58 (3H, m, CH₂), 2.70-2.82 (2H, m, CH₂), 3.86 (2H,s, ArCH₂S), 5.23 (2H, s, ArCH₂N), 6.66-6.72 (3H, m, ArH), 7.01-7.08 (2H,dd, J=2.2 & 6.7 Hz, ArH), 7.15 (2H, s, ArH), 7.92 (1H, s, C₂H₂N₃), 7.99(1H, s, C₂H₂N₃) and 8.18 (1H, bs, ArOH); ¹³C NMR (69.5 MHz, CDCl₃) δ17.2 (C), 34.5 (CH₂), 39.9 (CH₂), 41.1 (CH₂), 53.4 (CH₂), 116.3 (CH),123.5 (C), 123.9 (CH), 126.8 (CH), 128.2 (CH), 134.9 (C), 135.7 (CH),140.7 (C), 141.0 (C), 143.0 (CH), 151.7 (CH) and 156.8 (C); HPLC (90%CH₃CN in H₂O) t_(r)=1.876 (94.36%); LCMS (APCI), m/z 377.33 (M⁺+H,100%).

4-((3-((1H-1,2,4-triazol-1-yl)methyl)-5-(1-cyanocyclobutyl)benzyl)sulfanyl)phenylsulfamate (TJA01155, STX1731)

C₂₁H₂₁N₅O₃S₂ MW 455.55

Sulfamoyl chloride in toluene (0.60 M, 2.86 mL) was transferred to anr.b. flask and the solvent removed under vacuum at 30° C. On cooling awhite solid formed to which was added N,N-dimethylacetamide (1.5 mL) toform a colourless solution. TJA01151 (0.089 g, 0.236 mmol) was added andthe solution left to stir at room temperature under N_(2 (g)) for 72 h.The reaction mixture was then poured into distilled H₂O (30 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL×2). Dried overNa₂SO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a colourless viscous oil (0.058 g, 55%), R_(f): 0.40(dichloromethane/acetone 80:20); ¹H NMR (270 MHz, CDCl₃) δ 2.03-2.10(1H, m, CH₂), 2.42-2.61 (3H, m, CH₂), 2.77-2.82 (2H, m, CH₂), 4.03 (2H,s, ArCH₂SAr), 5.27 (2H, s, ArCH₂N), 6.59 (1H, s, ArH), 6.88 (2H, s,ArOSO₂NH₂), 7.11-7.33 (6H, m, ArH), 7.59 (1H, s, C₂H₂N₃) and 7.92 (1H,s, C₂H₂N₃); ¹³C NMR (69.5 MHz, CDCl₃) δ 17.2 (C), 34.6 (CH₂), 38.2(CH₂), 40.0 (CH₂), 53.2 (CH₂), 123.4 (CH), 124.0 (CH), 126.5 (CH), 126.8(CH), 132.5 (CH), 132.7 (C), 135.6 (C), 140.1 (C), 141.2 (C), 143.9(CH), 149.3 (C) and 151.2 (CH) (one overlapping signal); HPLC (90% CH₃CNin H₂O) t_(r)=1.827 (100%); LCMS (APCI), m/z 456.34 (M⁺+H, 100%), 377.39(M⁺−SO₂NH₂, 20%).Synthesis of STX1732

3-(1-Cyano-cyclopropyl)-5-methyl-benzoic acid methyl ester (TJA01099)

C₁₃H₁₃NO₂ MW 215.09

TJA01076 (1.50 g, 7.93 mmol) was loaded to a dry 50 mL r.b. flask whichwas subsequently purged with N_(2 (g)). To this was added dry DMF (10mL) and the solution cooled with stirring to 0° C. Sodium hydride (0.476g, 19.8 mmol) was carefully added resulting in a deep red coloration andevolution of gas. After 15 min at 0° C. 1,2-dibromoethane (0.820 mL,9.48 mmol) was added dropwise over 5 min. The reaction was allowed towarm to room temp. and left to stir for 1 h. Ethyl acetate (50 mL) wasadded to the reaction mixture and this was washed with distilled water(50 mL×4) and brine (50 mL). The organic layer was separated and driedover MgSO₄, filtered and solvent removed in vacuo. Column chromatography(hexane/EtOAc 70:30) eluted the title compound as a colourless oil(0.600 g, 35%), R_(f): 0.52 (hexane/EtOAc 75:25) ¹H NMR (270 MHz, CDCl₃)δ 1.40-1.45 (2H, dd, J=3.0 & 5.0 Hz, CH₂), 1.71-1.76 (2H, dd, J=3.7 &5.0 Hz, CH₂), 2.39 (3H, s, ArCH₃), 3.89 (3H, s, ArCO₂CH₃), 7.39 (1H, s,ArH), 7.62 (1H, s, ArH) and 7.75 (1H, s, ArH); ¹³C NMR (67.8 MHz, CDCl₃)δ 13.7 (CH₂), 18.4 (C), 21.4 (CH₃), 52.4 (CH₃), 122.4 (C), 123.4 (CH),129.6 (CH), 130.9 (C), 131.7 (CH), 136.5 (C), 139.2 (C) and 166.7 (C═O);HPLC (80% CH₃CN in H₂O) t_(r)=3.220 (98.85%); LCMS (APCI), m/z 216.09(M⁺+H, 100%), 157.92 ((M⁺+H)—CO₂CH₃, 92); Anal. Calcd. for C₁₃H₁₃NO₂: C72.54, H 6.09, N 6.51. Found: C 72.30, H 6.04, N 6.38%.

3-Bromomethyl-5-(1-cyano-cyclopropyl)-benzoic acid methyl ester(TJA01100)

C₁₃H₁₂BrNO₂ MW 294.15

To a solution of sodium bromate (2.53 g, 16.8 mmol) in distilled H₂O (12mL) was added TJA01099 (0.600 g, 2.79 mmol) in ethyl acetate (6 mL). Tothis clear mixture a solution of sodium hydrogen sulphate (2.02 g, 16.8mmol) in distilled H₂O (24 mL) was added drop wise with vigorousstirring over 15 min. The reaction mixture was stirred for a further 4 hat room temperature. The ethyl acetate was separated and diethyl ether(50 mL) added. This was then washed with saturated Na2SO_(3(aq)) (50 mL)distilled water (50 ml×2) and brine (50 mL). Dried over MgSO₄ andsolvent removed in vacuo. Column chromatography (hexane/ethyl acetate75:25) eluted the title compound as a white crystalline solid (0.760 g,96%), mp 78.3-79.8° C.; R_(f): 0.35 (hexane/ethyl acetate 75:25), c.f0.47 (TJA01091); ¹H NMR (270 MHz, CDCl₃) δ 1.44-1.49 (2H, dd, J=2.7 &5.2 Hz, CH₂), 1.76-1.79 (2H, dd, J=2.7 & 4.9 Hz, CH₂), 3.91 (3H, s,ArCO₂CH₃), 4.48 (2H, s, ArCH₂Br), 7.59 (1H, s, ArH), 7.76 (1H, s, ArH)and 7.97 (2H, s, ArH); ¹³C NMR (69.5 MHz, CDCl₃) δ 13.7 (CH₂), 18.7 (C),31.9 (CH₂), 52.6 (CH₃), 121.9 (C), 126.1 (CH), 129.5 (CH), 131.3 (CH),131.7 (C), 137.5 (C), 139.2 (C) and 165.9 (C═O); HPLC (80% CH₃CN in H₂O)t_(r)=3.138 (100%); LCMS (APCI), m/z 296.14 (⁸¹BrM⁺+H, 65%), (⁷⁹BrM⁺+H,70), 214.07 ((M⁺+H)−Br, 100).

3-(1-Cyano-cyclopropyl)-5-[1,2,4]triazol-1-ylmethyl-benzoic acid methylester (TJA01101)

C₁₅H₁₄N₄O₂ MW 282.11

TJA01100 (0.790 g, 2.69 mmol), 1,2,4-triazole (0.278 g, 0.403 mmol),potassium carbonate (0.372 g, 2.69 mmol), potassium iodide (0.026 g,0.158 mmol) and acetone (50 mL) were loaded to an r.b. flask. Withvigorous stirring this mixture was set to reflux (60° C.) for 16 h. Thereaction mixture was allowed to cool and acetone was removed in vacuo.The residues were taken up in ethyl acetate (50 mL) and washed withdistilled water (50 mL×3) and brine (50 mL). Dried over MgSO₄ andsolvent removed in vacuo to leave a dark brown syrup. Columnchromatography (hexane/ethyl acetate 75:25 then ethyl acetate) elutedthe title compound as a colourless viscous oil (0.444 g, 59%) R_(f):0.15 (ethyl acetate); ¹H NMR (300 MHz, CDCl₃) δ 1.39-1.45 (2H, dd, J=2.8& 5.8 Hz, CH₂), 1.76-1.80 (2H, dd, J=3.1 & 5.6 Hz, CH₂), 3.89 (3H, s,ArCO₂CH₃), 5.38 (2H, s, ArCH₂N), 7.49 (1H, s, ArH), 7.80 (1H, s, ArH),7.82 (1H, s, ArH), 7.98 (1H, s, C₂H₂N₃), and 8.12 (1H, s, C₂H₂N₃); ¹³CNMR (69.5 MHz, CDCl₃) δ (13.8 (CH₂), 18.8 (C), 52.7 (CH₃), 52.9 (CH₂),121.8 (C), 126.5 (CH), 128.3 (CH), 130.3 (CH), 131.9 (C), 136.2 (C),137.9 (C), 143.4 (CH), 152.7 (CH) and 165.8 (C═O); HPLC (90% CH₃CN inH₂O) t_(r)=1.935 (100%); LCMS (APCI), m/z 283.43 (M⁺+H, 100%), 214.33((M⁺+H)—C₂H₂N₃, 75); Anal. Calcd. for C₁₅H₁₄N₄O₂: C 63.82, H 5.00, N19.85 Found: C 63.50, H 5.01, N 19.80%.

1-(3-Hydroxymethyl-5-[1,2,4]triazol-1-ylmethyl-phenyl)-cyclopropanecarbonitrile(TJA01105)

C₁₄H₁₄N₄O MW 254.29

A 25 mL r.b. flask was loaded with TJA01101 (0.391 g, 1.39 mmol) andpolyethylene glycol 400 (5.0 g). The mixture was heated to 80° C. withstirring until a solution had formed. Sodium borahydride (0.157 g, 4.15mmol) was added carefully resulting in evolution of gas. The reactionmixture was stirred vigorously at 80° C. for 16 h. An extremely viscousglue formed that gradually dissolved in dichloromethane (50 mL) withheating (40° C.). This solution was washed with 1M HCl_((aq)) (10 mL)and then carefully neutralised with sodium bicarbonate. Washed withdistilled water (50 mL×4) and brine (50 mL), separated and dried overMgSO₄. Solvent removed in vacuo to leave a colourless viscous oil. Flashchromatography (20 g column, Flashmaster II, method TJA01097) eluted thetitle compound as a colourless viscous oil (0.274 g, 78%), R_(f): 0.10(ethyl acetate); ¹H NMR (300 MHz, CDCl₃) δ 1.36-1.40 (2H, dd, J=2.5 &5.3 Hz, CH₂), 1.70-1.75 (2H, dd, J=2.6 & 5.4 Hz, CH₂), 1.89-1.94 (1H, t,J=5.8 Hz, ArCH₂OH), 4.66-4.68 (2H, d, J=5.7 Hz, ArCH₂OH), 5.31 (2H, s,ArCH₂N), 7.12 (1H, s, ArH), 7.14 (1H, s, ArH), 7.24 (1H, s, ArH), 7.95(1H, s, NCHN), and 8.08 (1H, s, NCHN); ¹³C NMR (69.5 MHz, CDCl₃) δ 13.8(CH₂), 18.5 (CH₂), 53.3 (CH₂), 64.2 (CH₂), 122.3 (C), 124.2 (CH), 124.6(CH), 125.5 (CH), 135.8 (C), 137.4 (C), 143.3 (CH), 143.3 (C) and 152.3(CH); HPLC (90% CH₃CN in H₂O) t_(r)=1.791 (100%); LCMS (APCI), m/z255.09 (M⁺+H, 100%).

Methanesulfonic acid4-[3-(1-cyano-cyclopropyl)-5-[1,2,4]triazol-1-ylmethyl-benzylsulfanyl]-phenylester (TJA01147)

C₂₁H₂₀N₄O₃S₂ MW 440.54

A dry 5 mL r.b. flask purged with N_(2 (g)) was loaded with TJA01110(0.115 g, 0.472 mmol), TJA01105 (0.100 g, 0.393 mmol), TJA01130 (0.120,0.590 mmol), diisopropylethylamine (89.0 μL, 0.511 mmol) andpropionitrile (1.0 mL). The mixture was then set to stir at 93° C. After2 h a further 1 equivalent of TJA01110 (0.096 g, 0.393 mmol) anddiisopropylethylamine (68.4 μL, 0.393 mmol) were added. After 5 h thereaction was allowed to cool. Dichloromethane (20 mL) and distilledwater (20 mL) were added and the aqueous layer separated and extractedwith dichloromethane (20 mL×2). The organic fractions were combined andwashed with brine (20 mL), dried over MgSO₄ and solvent removed in vacuoto leave yellow residues. Column chromatography (ethyl acetate) elutedthe title compound as a yellow viscous oil (0.134 g, 77%), R_(f): 0.32(ethyl acetate); ¹H NMR (270 MHz, CDCl₃) δ 1.29-1.34 (2H, dd, J=5.0 &7.9 Hz, CH₂), 1.69-1.74 (2H, dd, J=5.0 & 7.6 Hz, CH₂), 3.16 (3H, s,ArOSO₂CH₃), 4.01 (2H, s, ArCH₂S), 5.27 (2H, s, ArCH₂N), 6.94 (1H, s,ArH), 7.05 (1H, s, ArH), 7.08 (1H, s, ArH), 7.14-7.28 (4H, dd, J=8.6 &27.9 Hz, ArH), 7.96 (1H, s, C₂H₂N₃) and 8.07 (1H, s, C₂H₂N₃); ¹³C NMR(69.5 MHz, CDCl₃) δ 13.6 (CH₂), 18.6 (C), 37.7 (CH₃), 39.2 (CH₂), 52.9(CH₂), 122.2 (C), 122.8 (CH), 124.1 (CH), 126.4 (CH), 127.5 (CH), 132.5(CH), 134.6 (C), 136.1 (C), 137.5 (C), 139.3 (C), 143.3 (CH), 147.9 (C)and 152.6 (CH); HPLC (90% CH₃CN in H₂O) t_(r)=1.853 (100%); LCMS (APCI),m/z 441.23 (M⁺+H, %).

1-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-((4-hydroxyphenylthio)methyl)phenyl)cyclopropanecarbonitrile(TJA01153)

C₂₀H₁₈N₄OS MW 362.45

TJA01147 (0.129 g, 0.293 mmol) was dissolved in THF (3 mL) and methanol(2 mL) to which 2M NaOH_((aq)) (0.73 mL) was added. The mixture was setto stir at room temp. for 12 h. THF was removed under reduced pressureand the residues taken up in ethyl acetate (20 mL) and washed with 2MKHSO_(4 (aq)) (20 mL), distilled water (20 mL×2) and brine (20 mL). Theorganic layer was then dried over MgSO₄ and solvent removed underreduced pressure to leave a colourless viscous oil. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a white solid (0.076 g, 72%), R_(f): 0.25 (dichloromethane/acetone80:20); ¹H NMR (270 MHz, CDCl₃) δ 1.31-1.35 (2H, dd, J=5.3 & 8.2 Hz,CH₂), 1.68-1.73 (2H, dd, J=5.2 & 7.6 Hz, CH₂), 3.83 (2H, s, ArCH₂S),5.21 (2H, s, ArCH₂N), 6.65-6.70 (3H, m, ArH), 6.99-7.04 (3H, m, ArH),7.11 (1H, s, ArH), 7.91 (1H, s, C₂H₂N₃), 7.99 (1H, s, C₂H₂N₃) and 8.12(1H, s, ArOH); ¹³C NMR (69.5 MHz, CDCl₃) δ 13.6 (CH₂), 18.4 (C), 41.1(CH₂), 53.3 (CH₂), 116.5 (CH), 122.2 (C), 123.6 (C), 124.5 (CH), 126.5(CH), 127.8 (CH), 134.9 (C), 135.8 (CH), 137.4 (C), 140.7 (C), 143.0(CH), 151.7 (CH) and 156.7 (C); HPLC (90% CH₃CN in H₂O) t_(r)=1.843(93.71%); LCMS (APCI), m/z 363.35 (M⁺+H, %).

4-((3-((1H-1,2,4-triazol-1-yl)methyl)-5-(1-cyanocyclopropyl)benzyl)sulfanyl)phenylsulfamate (TJA01156, STX1732)

C₂₀H₁₉N₅O₃S₂ MW 441.53

Sulfamoyl chloride in toluene (0.60 M, 1.42 mL) was transferred to a 10mL r.b. flask and the solvent removed under vacuum at 30° C. On coolinga white solid formed to which was added N,N-dimethylacetamide (1.5 mL)to form a colourless solution. TJA01153 (0.062 g, 0.171 mmol) was addedand the solution left to stir at room temperature under N_(2 (g)) for 72h. The reaction mixture was then poured into distilled H₂O (30 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL×2). Dried overMgSO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a colourless viscous oil (0.055 g, 73%), R_(f): 0.22(dichloromethane/acetone 80:20); ¹H NMR (270 MHz, CDCl₃) δ 1.39-1.44(2H, dd, J=5.0 & 7.9 Hz, CH₂), 1.74-1.79 (2H, dd, J=5.1 & 7.8 Hz, CH₂),4.01 (2H, s, ArCH₂SAr), 5.21 (2H, s, ArCH₂N), 6.55 (1H, s, ArH), 6.88(2H, s, ArOSO₂NH₂), 7.03 (1H, s, ArH), 7.11-7.21 (5H, m, ArH), 7.54 (1H,s, C₂H₂N₃) and 7.85 (1H, s, C₂H₂N₃); ¹³C NMR (69.5 MHz, CDCl₃) δ 13.7(CH₂), 18.7 (C), 38.3 (CH₂), 53.2 (CH₂), 122.2 (C), 123.4 (CH), 123.9(CH), 126.5 (CH), 126.6 (CH), 132.4 (CH), 132.9 (C), 135.6 (C), 137.6(C), 140.1 (C), 142.9 (CH), 149.3 (C) and 151.1 (CH); HPLC (90% CH₃CN inH₂O) t_(r)=1.791 (100%); LCMS (APCI), m/z 442.30 (M⁺+H, 100%), 363.41(M⁺−SO₂NH₂, 35%).Synthesis of STX1761

2-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-((4-(benzyloxy)phenoxy)methyl)phenyl)-2-methylpropanenitrile(TJA01159)

C₂₇H₂₆N₄O₂ MW 438.52

A dry 25 mL flask was loaded with TJA01097 (0.150 g, 0.586 mmol) and4-(benzyloxy)phenol (0.098 g, 0.488 mmol) and purged with N_(2 (g)).Anhydrous dichloromethane (3 mL) was added to form a clear solution andto this was added triphenylphosphine polystyrene (0.586 g, 0.586 mmol)and the mixture cooled to 0° C. Diethylazodicarboxylate (92.3 μL, 0.586mmol) was added dropwise and the reaction allowed to warm to roomtemperature and left to stir under N_(2 (g)) for 16 h. The reactionmixture was then filtered to remove the polystyrene which was washedwith dichloromethane (6×25 mL). These washings were combined with thefiltrate and washed with distilled H₂O (2×25 mL) and brine (25 mL) thendried over MgSO₄ and solvent removed in vacuo. Column chromatography(ethyl acetate) eluted the title compound as a light yellow solid (0.175g, 82%), R_(f): 0.39 (ethyl acetate); ¹H NMR (270 MHz, CDCl₃) δ 1.70(6H, s, ArC(CH₃)₂CN), 4.98 (2H, s, ArCH₂O), 5.01 (2H, s, ArCH₂O), 5.37(2H, s, ArCH₂N), 6.84-6.92 (4H, m, ArH), 7.29-7.49 (8H, m, ArH), 7.99(1H, s, C₂H₂N₃) and 8.10 (1H, s, C₂H₂N₃); ¹³C NMR (69.5 MHz, CDCl₃) δ29.2 (CH₃), 37.2 (C), 53.3 (CH₂), 70.1 (CH₂), 70.7 (CH₂), 115.8 (CH),115.9 (CH), 124.1 (C), 124.3 (CH), 124.5 CH), 126.4 (CH), 127.6 (CH),128.0 (CH), 128.7 (CH), 136.1 (C), 137.2 (C), 139.4 (C), 142.9 (C),143.3 (CH), 152.5 (CH), 152.8 (C) and 153.5 (C); HPLC (90% CH₃CN in H₂O)t_(r)=2.406 (94.36%); LCMS (APCI), m/z 439.47 (M⁺+H, 100%).

2-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-((4-hydroxyphenoxy)methyl)phenyl)-2-methylpropanenitrile(TJA01162)

C₂₀H₂₀N₄O₂ MW 348.40

TJA01159 (0.100 g, 0.228 mmol) was dissolved in THF (2.5 mL) and MeOH(2.5 mL) in an r.b. flask to which was added 5% Pd/C (0.010 g) to form ablack suspension on vigorous stirring. The flask was evacuated and backfilled with H_(2 (g)) via a balloon (×3) and then left to stir for 8 hwhen more 5% Pd/C (0.010 g) was added. The reaction was then left tostir for a further 16 h. The reaction mixture was filtered throughcelite which was subsequently washed with THF (30 mL×2). Solvent wasremoved in vacuo to leave a brown residue. Column chromatography (ethylacetate) eluted the title compound as a colourless viscous oil (0.060 g,76%), R_(f): 0.30 (ethyl acetate); ¹H NMR (270 MHz, CDCl₃) δ 1.70 (6H,s, ArC(CH₃)₂CN), 4.97 (2H, s, ArCH₂O), 5.17 (1H, s, ArOH), 5.37 (2H, s,ArCH₂N), 6.73-6.82 (4H, m, ArH), 7.22 (1H, s, ArH), 7.33 (1H, s, ArH),7.49 (1H, s, ArH), 7.99 (1H, s, C₂H₂N₂) and 8.10 (1H, s, C₂H₂N₂); HPLC(90% CH₃CN in H₂O) t_(r)=1.870 (97.88%); LCMS (APCI), m/z 349.43 (M⁺+H,100%).

4-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-(2-cyanopropan-2-yl)benzyl)phenoxysulfamate (TJA01165, STX1761)

C₂₀H₂₁N₅O₄S MW 427.48

Sulfamoyl chloride in toluene (0.60 M, 1.41 mL) was transferred to a 10mL r.b. flask and the solvent removed under vacuum at 30° C. On coolinga white solid formed to which was added N,N-dimethylacetamide (1.5 mL)to form a colourless solution. TJA01162 (0.059 g, 0.169 mmol) was addedand the solution left to stir at room temperature under N_(2 (g)) for 16h. The reaction mixture was then poured into distilled H₂O (30 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL×2). Dried overMgSO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a white solid (0.049 g, 68%), R_(f): 0.51 (dichloromethane/acetone80:20); ¹H NMR (400 MHz, DMSO-d₆) δ 1.68 (6H, s, ArC(CH₃)₂CN), 5.11 (2H,s, ArCH₂OAr), 5.48 (2H, s, ArCH₂N), 7.05-7.21 (4H, dd, J=8.8 & 53.2 Hz,AA′BB′), 7.30 (1H, s, ArH), 7.46 (1H, s, ArH), 7.57 (1H, s, ArH), 7.91(2H, s, ArOSO₂NH₂), 8.00 (1H, s, C₂H₂N₂) and 8.69 (1H, s, C₂H₂N₂); ¹³CNMR (100.5 MHz, DMSO-d₆) δ 18.3, 26.6, 41.8, 59.3, 105.6, 113.4, 114.2,114.4, 116.5, 127.4, 128.2, 132.2, 133.8, 134.4, 141.9 and 146.6 (oneoverlapping signal); HPLC (70% CH₃CN in H₂O) t_(r)=2.441 (100%); LCMS(APCI), m/z 428.39 (M⁺+H, 100%).Synthesis of STX1794

3-Chloro-4-methanesulfonyloxy-benzenesulfonic acid sodium salt(TJA01127)

C₇H₆ClNaO₆S₂ MW 308.69

SO₃ ⁻Na⁺

4-hydroxy-3-chlorobenzenesulfonic acid sodium salt (11.53 g, 50.0 mmol)and sodium hydroxide (2.00 g, 50.0 mmol) were dissolved in distilledwater (50 mL) and the solution cooled to 0° C. Methane sulfonyl chloride(4.25 mL, 55.0 mmol) was added dropwise with stirring and the mixturethen allowed to warm to room temp. and left for 2 h. Brine (20 mL) wasadded and the solution left to stand for 1 h with the formation of whitecrystalline solid. The solids were filtered, recrystallised (brine), anddried under vacuum to give the title compound as a white crystallinesolid (9.40 g, 61%), mp>250° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 3.31 (3H,s, ArOSO₂CH₃), 7.48-7.51 (1H, d, J=8.4 Hz, ArH), 7.59-7.62 (1H, dd,J=2.2 & 8.4 Hz, ArH) and 7.69-7.72 (1H, d, J=2.2 Hz, ArH); ¹³C NMR (69.5MHz, DMSO-d₆) δ 39.1 (CH₃), 124.4 (CH), 126.3 (C), 126.5 (CH), 128.3(CH), 145.3 (C) and 148.8 (C).

Methanesulfonic acid 2-chloro-4-chlorosulfonyl-phenyl ester (TJA01128)

C₇H₆Cl2O₅S₂ MW 305.16

Thionyl chloride (30 mL) was cooled to 0° C. Cautiously, with stirring,TJA01127 (8.60 g, 28.0 mmol) was added followed by DMF (0.5 mL). Thereaction mixture was subsequently heated to reflux (79° C.) for 1 h (oruntil evolution of gas has ceased) and then cooled. Thionyl chloride wasremoved in vacuo and the resulting yellow residues were taken up indichloromethane (50 mL) and distilled water (50 mL) carefully added. Theorganic layer was separated and washed with distilled water (50 mL×2)and brine (50 mL), dried over MgSO₄ and solvent removed in vacuo toleave yellow residues. Recystallisation (dichloromethane/hexane) gavethe title compound as a white crystalline solid (7.10 g, 84%), ¹H NMR(300 MHz, CDCl₃) δ 3.37 (3H, s, ArOSO₂CH₃), 7.70-7.73 (1H, d, J=8.8 Hz,ArH), 7.99-8.03 (1H, dd, J=2.4 & 8.8 Hz, ArH) and 8.19-8.20 (1H, d,J=2.4 Hz, ArH); ¹³C NMR (69.5 MHz, CDCl₃) δ 39.6 (CH₃), 125.6 (CH),127.2 (CH), 128.8 (C), 129.9 (CH), 143.1 (C) and 149.9 (C); HPLC (90%CH₃CN in H₂O) t_(r)=2.489 (99.62%);.

Methanesulfonic acid 2-chloro-4-mercapto-phenyl ester (TJA01129)

C₇H₇ClO₃S₂ MW 238.71

A 50 mL r.b. flask was loaded with red phosphorus powder (0.630 g, 20.5mmol), iodine (0.035 g, 0.137 mmol) and acetic acid (7 mL). CautiouslyTJA01128 (2.50 g, 8.19 mmol) was added and the reaction mixture then setto reflux (118° C.) for 2 h. Distilled water (1.5 mL) was added and themixture left to reflux for a further 1 h. Reaction allowed to cool.Chloroform (30 mL) and distilled water (30 mL) were added. The organiclayer was separated and washed with distilled water (30 mL×3) and brine(30 mL). Dried over MgSO₄, filtered and solvent removed in vacuo. Columnchromatography (ethyl acetate/hexane 50:50) eluted the title compound asa colourless viscous oil (1.68 g, 87%), R_(f): 0.71 (ethyl acetate); ¹HNMR (300 MHz, CDCl₃) δ 3.24 (3H, s, ArOSO₂CH₃), 3.56 (1H, s, ArSH),7.18-7.22 (1H, dd, J=2.3 & 8.5 Hz, ArH), 7.30-7.33 (1H, d, J=8.1 Hz,ArH) and 7.39-7.40 (1H, d, J=2.2 Hz, ArH); ¹³C NMR (69.5 MHz, CDCl₃) δ38.7 (CH₃), 125.1 (CH), 127.4 (C), 128.9 (CH), 130.9 (CH), 132.1 (C) and143.2 (C); HPLC (90% CH₃CN in H₂O) t_(r)=1.811 (92.46%); LCMS (APCI),m/z 239.01 (³⁷ClM⁺−H, 10%), 239.01 (³⁵ClM⁺−H, 30).

4-((3-((1H-1,2,4-triazol-1-yl)methyl)-5-(1-cyanocyclobutyl)benzyl)sulfanyl)-2-chlorophenylmethanesulfonate (TJA01170)

C₂₂H₂₁ClN₄O₃S₂ MW 489.01

A dry 5 mL r.b. flask purged with N_(2 (g)) was loaded with TJA01110(0.107 g, 0.447 mmol), TJA01104 (0.100 g, 0.373 mmol), TJA01129 (0.133,0.559 mmol), diisopropylethylamine (84.5 μL, 0.485 mmol) andpropionitrile (1.0 mL). The mixture was then set to stir at 93° C. underN_(2 (g)). After 3 h a further 1 equivalent of TJA01110 (0.091 g, 0.373mmol), TJA01129 (0.087, 0.373) and diisopropylethylamine (65.0 μL, 0.373mmol) were added. After 20 h the reaction was allowed to cool.Dichloromethane (20 mL) and distilled water (20 mL) were added and theaqueous layer separated and extracted with dichloromethane (20 mL×2).The organic fractions were combined and washed with brine (20 mL), driedover MgSO₄ and solvent removed in vacuo to leave yellow residues. Columnchromatography (ethyl acetate) eluted the title compound as a yellowviscous oil (0.054 g, 30%), R_(f): 0.43 (ethyl acetate); ¹H NMR (270MHz, CDCl₃) δ 1.96-2.08 (1H, m, CH₂), 2.31-2.58 (3H, m, CH₂), 2.70-2.83(2H, m, CH₂), 3.24 (3H, s, ArOSO₂CH₃), 4.05 (2H, s, ArCH₂S), 5.31 (2H,s, ArCH₂N), 7.00 (1H, s, ArH), 7.08-7.32 (5H, m, ArH), 7.96 (1H, s,C₂H₂N₃) and 8.10 (1H, s, C₂H₂N₃); ¹³C NMR (69.5 MHz, CDCl₃) δ 17.2 (C),34.7 (CH₂), 38.8 (CH₂), 38.9 (CH₃), 40.0 (CH₂), 53.0 (CH₂), 123.9 (C),124.4 (CH), 124.9 (CH), 126.4 (CH), 127.3 (C), 127.7 (CH), 129.9 (CH),132.0 (CH), 136.2 (C), 136.3 (C), 138.7 (C), 141.3 (C), 143.4 (CH),143.9 (C) and 152.5 (CH); HPLC (90% CH₃CN in H₂O) t_(r)=3.086 (99.60%);LCMS (APCI), m/z 489.27 (M⁺+H, 100%).

1-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-((3-chloro-4-hydroxyphenylthio)methyl)phenyl)cyclobutanecarbonitrile(TJA01172)

C₂₁H₁₉ClN₄OS MW 410.92

TJA01170 (0.050 g, 0.102 mmol) was dissolved in THF (2 mL) and methanol(2 mL) to which 2M NaOH_((aq)) (0.260 mL) was added. The mixture was setto stir at room temp. for 3 h. THF was removed under reduced pressureand the residues taken up in ethyl acetate (20 mL) and washed with 2MKHSO_(4 (aq)) (20 mL), distilled water (20 mL×2) and brine (20 mL). Theorganic layer was then dried over MgSO₄ and solvent removed underreduced pressure to leave the title compound as a light yellow viscousoil (0.039 g, 99%), R_(f): 0.25 (dichloromethane/acetone 80:20); ¹H NMR(270 MHz, CDCl₃) δ 1.92-2.12 (1H, m, CH₂), 2.31-2.62 (3H, m, CH₂),2.65-2.88 (2H, m, CH₂), 3.89 (2H, s, ArCH₂SAr), 5.28 (2H, s, ArCH₂N),6.81-7.28 (6H, m, ArH), 7.99 (1H, s, C₂H₂N₃), 7.99 (1H, s, C₂H₂N₃) and8.06 (1H, bs, ArOH); ¹³C NMR (69.5 MHz, CDCl₃) δ 17.2 (C), 34.6 (CH₂),39.9 (CH₂), 40.9 (CH₂), 53.3 (CH₂), 117.1 (CH), 120.6 (C), 124.0 (C),124.1 (CH), 125.2 (C), 126.6 (CH), 127.9 (CH), 133.8 (CH), 134.6 (CH),135.5 (C), 140.1 (C), 141.0 (C), 143.1 (CH), 151.9 (CH) and 152.3 (C);HPLC (70% CH₃CN in H₂O) t_(r)=2.902 (85.05%); LCMS (APCI), m/z 411.45(M⁺+H, 100%).

4-((3-((1H-1,2,4-triazol-1-yl)methyl)-5-(1-cyanocyclobutyl)benzyl)sulfanyl)-2-chlorophenylsulfamate (TJA01176, STX1794)

C₂₁H₂₀ClN₅O₃S₂ MW 490.00

Sulfamoyl chloride in toluene (0.60 M, 2.86 mL) was transferred to an 10mL r.b. flask and the solvent removed under vacuum at 30° C. On coolinga white solid formed to which was added N,N-dimethylacetamide (1.5 mL)to form a colourless solution. TJA01172 (0.039 g, 0.100 mmol) was addedand the solution left to stir at room temperature under N_(2 (g)) for 60h. The reaction mixture was then poured into distilled H₂O (30 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL×2). Dried overMgSO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a white amorphous solid (0.037 g, 77%), R_(f): 0.28(dichloromethane/acetone 80:20); ¹H NMR (270 MHz, CDCl₃) δ 2.04-2.13(1H, m, CH₂), 2.44-2.67 (3H, m, CH₂), 2.80-2.87 (2H, m, CH₂), 4.06 (2H,s, ArCH₂SAr), 5.28 (2H, s, ArCH₂N), 6.56 (1H, s, ArH), 6.89-6.97 (1H,dd, J=2.2 & 8.6 Hz, ArH), 7.10 (2H, bs, ArOSO₂NH₂), 7.23-7.30 (4H, m,ArH), 7.62 (1H, s, C₂H₂N₃) and 7.86 (1H, s, C₂H₂N₃); HPLC (70% CH₃CN inH₂O) t_(r)=2.557 (99.59%); LCMS (APCI), m/z 492.29 (³⁷ClN⁺+H, 40%),490.27 (³⁵ClM⁺+H, 100).Synthesis of STX1795

3-Bromo-4-hydroxy-benzenesulfonic acid sodium salt (TJA01144)

C₆H₄BrNaO₄S MW 275.05

2-Bromophenol (20.0 g, 116 mmol) was added with stirring to a 250 mLr.b. flask containing sulphuric acid, 98%, (50 mL) and the mixture leftto stir at 100° C. for 2 h. The reaction mixture was allowed to cool andthen poured into brine (100 mL) and left to stand for 1 h. White solidsformed and were collected via filtration, recrystallised (brine) anddried under reduced pressure to give the title compound as a whitecrystalline solid (13.7 g, 43%), mp>250° C.; ¹H NMR (270 MHz, DMSO-d₆) δ6.92-6.95 (1H, d, J=8.4 Hz, ArH), 7.38-7.42 (1H, dd, J=2.0 & 8.4 Hz,ArH), 7.64-7.65 (1H, d, J=2.0 Hz, ArH) and 10.40-10.74 (1H, bs, ArOH);¹³C NMR (69.5 MHz, DMSO-d₆) δ 108.5 (C), 115.9 (CH), 126.7 (CH), 130.8(CH), 141.1 (C) and 154.9 (C).

Sodium 3-bromo-4-methanesulfonoylbenzenesulfonate (TJA01148)

C₇H₆BrNaO₆S₂ MW 353.14

TJA01144 (13.8 g, 50.0 mmol) and sodium hydroxide (2.00 g, 50.0 mmol)were dissolved in distilled water (50 mL) and the solution cooled to 0°C. Methane sulfonyl chloride (4.25 mL, 55.0 mmol) was added dropwisewith stirring and the mixture then allowed to warm to room temp. andleft for 2 h. Brine (20 mL) was added and the solution left to stand for1 h with the formation of white crystalline solid. The solids werefiltered, recrystallised (brine), and dried under vacuum to give thetitle compound as a white crystalline solid (7.74 g, 44%), mp>250° C.;¹H NMR (270 MHz, DMSO-d₆) δ 3.56 (3H, s, ArOSO₂CH₃), 7.49-7.53 (1H, d,J=12.3 Hz, ArH), 7.64-7.68 (1H, dd, J=2.2 & 8.4 Hz, ArH) and 7.85-7.88(1H, d, J=6.2 Hz, ArH); ¹³C NMR (69.5 MHz, DMSO-d₆) δ 39.4 (CH₃), 115.8(C), 124.1 (CH), 127.1 (CH), 131.3 (CH), 146.5 (C) and 148.9 (C).

Methanesulfonic acid 2-bromo-4-chlorosulfonyl-phenyl ester (TJA01168)

C₇H₆BrClO₅S₂ MW 349.61

Thionyl chloride (30 mL) was cooled to 0° C. Cautiously, with stirring,TJA01148 (7.70 g, 21.8 mmol) was added followed by DMF (0.5 mL). Thereaction mixture was subsequently heated to reflux (79° C.) for 1 h (oruntil evolution of gas has ceased) and then cooled. Thionyl chloride wasremoved in vacuo and the resulting yellow residues were taken up indichloromethane (50 mL) and distilled water (50 mL) carefully added. Theorganic layer was separated and washed with distilled water (50 mL×2)and brine (50 mL), dried over MgSO₄ and solvent removed in vacuo toleave yellow residues. Recystallisation (dichloromethane/hexane) gavethe title compound as a white crystalline solid (2.47 g, 32%), ¹H NMR(270 MHz, CDCl₃) δ 3.36 (3H, s, ArOSO₂CH₃), 7.67-7.71 (1H, d, J=8.7 Hz,ArH), 8.02-8.06 (1H, dd, J=2.5 & 8.7 Hz, ArH) and 8.31-8.32 (1H, d,J=2.2 Hz, ArH); ¹³C NMR (69.5 MHz, CDCl₃) δ 39.8 (CH₃), 117.4 (C), 125.1(CH), 127.8 (CH), 132.8 (CH), 143.2 (C) and 151.2 (C); HPLC (90% CH₃CNin H₂O) t_(r)=2.108 (93.16%).

2-Bromo-4-mercaptophenyl methanesulfonate (TJA01169)

C₇H₇BrO₃S₂ MW 283.16

A 50 mL r.b. flask was loaded with red phosphorus powder (0.500 g, 16.1mmol), iodine (0.027 g, 0.107 mmol) and acetic acid (7 mL). CautiouslyTJA01168 (2.25 g, 6.44 mmol) was added and the reaction mixture then setto reflux (118° C.) for 2 h. Distilled water (1.5 mL) was added and themixture left to reflux for a further 1 h. Reaction allowed to cool.Chloroform (30 mL) and distilled water (30 mL) were added. The organiclayer was separated and washed with distilled water (30 mL×3) and brine(30 mL). Dried over MgSO₄, filtered and solvent removed in vacuo. Columnchromatography (ethyl acetate/hexane 50:50) eluted the title compound asa yellow viscous oil (1.45 g, 80%), R_(f): 0.41 (ethyl acetate); ¹H NMR(270 MHz, CDCl₃) δ 3.23 (3H, s, ArOSO₂CH₃), 3.54 (1H, s, ArSH),7.18-7.24 (1H, dd, is J=2.2 & 8.6 Hz, ArH), 7.28-7.31 (1H, d, J=8.4 Hz,ArH) and 7.52-7.53 (1H, d, J=2.2 Hz, ArH); ¹³C NMR (69.5 MHz, CDCl₃) δ38.9 (CH₃), 116.3 (C), 124.7 (CH), 129.7 (CH), 132.3 (C), 133.8 (CH) and144.5 (C); HPLC (90% CH₃CN in H₂O) t_(r)=2.604 (77.34%); LCMS (APCI),m/z 283.10 (⁸¹BrM⁺−H, 50%), 281.10 (⁷⁹BrM⁺−H, 65).

Methanesulfonic acid2-bromo-4-[3-(cyano-dimethyl-methyl)-5-[1,2,4]triazol-1-ylmethyl-benzylsulfanyl]-phenylester (TJA01171)

C₂₁H₂₁BrN₄O₃S₂ MW 521.45

TJA01110 (0.114 g, 0.470 mmol) was added to a mixture of TJA01097 (0.100g, 0.390 mmol), TJA01169 (0.161 g, 0.570 mmol), diisopropylethylamine(88.0 μL, 0.510 mmol) and propionitrile (1.0 mL) in a dry 5 mL r.b.flask purged with N_(2 (g)). The mixture was then set to stir at 92° C.After 2 h a further 1 equivalent of TJA01110 (0.094 g, 0.390 mmol),diisopropylethylamine (67.8 μL, 0.390 mmol) and TJA01169 (0.107 g, 0.380mmol) was added. After 6 h this addition was repeated. After 20 h thereaction was allowed to cool. Dichloromethane (20 mL) and distilledwater (20 mL) were added and the aqueous layer separated and extractedwith dichloromethane (20 mL×2). The organic fractions were combined andwashed with brine (20 mL), dried over MgSO₄ and solvent removed in vacuoto leave yellow residues. Column chromatography (ethyl acetate) elutedthe title compound as a yellow viscous oil (0.152 g, 75%), R_(f): 0.45(dichloromethane/acetone 80:20). ¹H NMR (270 MHz, CDCl₃) δ 1.62 (6H, s,ArC(CH₃)₂CN), 3.25 (3H, s, ArOSO₂CH₃), 4.04 (2H, s, ArCH₂SAr), 5.31 (2H,s, ArCH₂N), 7.00 (1H, s, ArH), 7.16-7.31 (4H, m, ArH), 7.45-7.46 (1H, d,J=2.2 Hz, ArH), 7.24 (1H, s, ArH), 7.97 (1H, s, C₂H₂N₃) and 8.10 (1H, s,C₂H₂N₃); ¹³C NMR (69.5 MHz, CDCl₃) δ 29.1 (CH₃), 37.1 (C), 39.0 (CH₂),39.1 (CH₃), 52.1 (CH₂), 116.3 (C), 123.8 (CH), 124.0 (C), 124.6 (CH),126.0 (CH), 127.7 (CH), 130.8 (CH), 135.2 (CH), 136.3 (C), 138.8 (C),143.0 (C), 143.4 (CH), 145.2 (C) and 152.2 (CH) (one overlappingsignal); HPLC (90% CH₃CN in H₂O) t_(r)=1.985 (99.16%); LCMS (APCI), m/z523.32 (⁸¹BrM⁺+H, 100%), 521.31 (⁷⁹BrM⁺+H, 85).

2-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-((3-bromo-4-hydroxyphenylthio)methyl)phenyl)-2-methylpropanenitrile(TJA01174)

C₂₀H₁₉BrN₄OS MW 443.36

TJA01171 (0.150 g, 0.288 mmol) was dissolved in THF (3 mL) and methanol(2 mL) to which 2M NaOH_((aq)) (0.719 mL) was added. The mixture was setto stir at room temp. for 4 h. THF was removed under reduced pressureand the residues taken up in ethyl acetate (20 mL) and washed with 2MKHSO_(4 (aq)) (20 mL), distilled water (20 mL×2) and brine (20 mL). Theorganic layer was then dried over MgSO₄ and solvent removed underreduced pressure. Column chromatography (dichloromethane/acetone 80:20)eluted the title compound as a colourless viscous oil (0.087 g, 68%),R_(f): 0.41 (dichloromethane/acetone 80:20). ¹H NMR (270 MHz, CDCl₃) δ1.63 (6H, s, ArC(CH₃)₂CN), 3.90 (2H, s, ArCH₂SAr), 5.29 (2H, s, ArCH₂N),6.75 (1H, bs, ArOH), 6.83-6.87 (2H, m, ArH), 7.06-7.09 (1H, dd, J=2.2 &8.4 Hz, ArH), 7.14 (1H, s, ArH), 7.24 (1H, s, ArH), 7.30-7.31 (1H, d,J=2.2 Hz, ArH), 7.99 (1H, s, C₂H₂N₃) and 8.04 (1H, s, C₂H₂N₃); ¹³C NMR(69.5 MHz, CDCl₃) δ 29.1 (CH₃), 37.1 (C), 41.0 (CH₂), 53.3 (CH₂), 110.3(C), 116.8 (C), 123.6 (CH), 124.1 (C), 126.1 (CH), 127.9 (CH), 134.6(CH), 135.7 (C), 137.2 (CH), 140.0 (C), 142.7 (C), 143.2 (CH), 152.2(CH) and 152.9 (C) (one overlapping signal); HPLC (90% CH₃CN in H₂O)t_(r)=2.063 (78.41%); LCMS (APCI), m/z 445.32 (⁸¹BrM⁺+H, 89%), 443.31(⁷⁹BrM⁺+H, 100).

4-((3-((1H-1,2,4-triazol-1-yl)methyl)-5-(2-cyanopropan-2-yl)benzyl)sulfanyl)-2-bromophenylsulfamate (TJA01179, STX1795)

C₂₀H₂₀BrN₅O₃S₂ MW 522.44

Sulfamoyl chloride in toluene (0.60 M, 1.35 mL) was transferred to an 10mL r.b. flask and the solvent removed under vacuum at 30° C. On coolinga white solid formed to which was added N,N-dimethylacetamide (1.5 mL)to form a colourless solution. TJA01174 (0.072 g, 0.162 mmol) was addedand the solution left to stir at room temperature under N_(2 (g)) for 14h. The reaction mixture was then poured into distilled H₂O (30 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL×2). Dried overMgSO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a white solid (0.082 g, 96%), R_(f): 0.39 (dichloromethane/acetone80:20). ¹H NMR (270 MHz, CDCl₃) δ 1.63 (6H, s, ArC(CH₃)₂CN), 4.06 (2H,s, ArCH₂SAr), 5.28 (2H, s, ArCH₂N), 6.54 (1H, s, ArH), 6.97-7.01 (1H,dd, J=2.2 & 8.6 Hz, ArH), 7.09 (2H, bs, ArOSO₂NH₂), 7.25-7.31 (2H, m,ArH), 7.35 (1H, s, ArH), 7.40-7.41 (1H, d, J=2.2 Hz, ArH), 7.63 (1H, s,C₂H₂N₃) and 7.87 (1H, s, C₂H₂N₃); HPLC (70% CH₃CN in H₂O) t_(r)=2.402(100%); LCMS (APCI), m/z 524.27 (⁸¹BrM⁺+H, 100%), 522.25 (⁷⁹BrM⁺+H, 80).Synthesis of STX1833

1-Bromo-3-bromomethyl-5-methylbenzene (TJA01023)

C₈H₈Br₂ MW 263.96

To a solution of sodium bromate (24.4 g, 162 mmol) in distilled H₂O (40mL) was added 5-bromo-m-xylene (10.0 g, 54.0 mmol) in cyclohexane (108mL). To this clear mixture a solution of sodium hydrogen sulphate (30.8g, 162 mmol) in distilled H₂O (81 mL) was added drop wise with vigorousstirring over 60 min. The reaction mixture was stirred for a further 3 hat room temperature. The ethyl acetate was separated and diethyl ether(100 mL) added. This was then washed with saturated Na2SO_(3(aq)) (100mL), distilled water (100 ml×2) and brine (100 mL). Dried over Na₂SO₄and solvent removed in vacuo to leave a clear syrup. Columnchromatography (hexane) eluted the title compound as a clear oil thatcrystallised on standing to give a white crystalline solid that was usedwithout further purification (8.45 g, 60%); R_(f) 0.52 (hexane), c.f0.52 (dibromobenzylbromide), 0.45 (1,5-dibenzylbromide), 0.6(5-bromo-m-xylene).

¹H NMR (270 MHz, CDCl₃) δ 2.31 (3H, s, ArCH₃), 4.38 (2H, s, ArCH₂Br),7.11 (1H, s, ArH), 7.25 (1H, s, ArH) and 7.32 (1H, s, ArH); HPLC (60%CH₃CN in H₂O) t_(r)=3.877 (67%), 4.644 (31%, dibromobenzylbromide).

(3-Bromo-5-methyl-phenyl)acetonitrile (TJA01029)

C₉H₈BrN MW 210.07

TJA01023 (11.3 g, 42.7 mmol), potassium cyanide (3.34 g, 51.2 mmol) andtetrabutylammonium bromide (0.700 g, 2.10 mmol) were loaded to an r.b.flask together with dichloromethane (60 mL) and distilled water (15 mL).With vigorous stirring the reaction mixture was set to reflux (45° C.)for 24 h. On cooling the organic fraction was separated and washed withdistilled water (50 mL×2) and brine (50 mL×2) then dried over Na₂SO₄ andsolvent removed in vacuo to leave a red/orange oil. Columnchromatography initially eluting with hexane separated thedibromobenzylbromide impurity. Further elution withhexane/dichloromethane (50:50) gave the title compound as a clear yellowoil (6.63 g, 74%), R_(f) 0.54 (hexane/dichloromethane 50:50) ¹H NMR (270MHz, CDCl₃) δ 2.31 (3H, s, ArCH₃), 3.66 (2H, s, ArCH₂CN), 7.06 (1H, s,ArH), 7.25 (1H, s, ArH) and 7.27 (1H, s, ArH); ¹³C NMR (100.5 MHz,CDCl₃) δ 21.1, 23.2, 117.4, 122.8, 127.4, 128.1, 131.7, 131.9 and 141.1;HPLC (80% CH₃CN in H₂O) t_(r)=2.278 (72.5%); LCMS (APCI), m/z 211.78(⁸¹BrM⁺, 53%), 209.78 (⁷⁹BrM⁺, 55), 184.83 (⁸¹BrM⁺−CN, 80), 182.83(⁷⁹BrM⁺−CN, 76).

2-(3-Bromo-5-methylphenyl)-2-methyl-propionitrile (TJA01035)

C₁₁H₁₂BrN MW 238.13

To a dry r.b. flask purged with N_(2 (g)) was added TJA01029 (6.00 g,28.6 mmol) and dry THF (20 mL). With stirring this was cooled via anice-water bath and NaH (1.71 g, 71.4 mmol) was added gradually and thenleft to stir at 0° C. under N_(2 (g)) for 15 min. Iodomethane (3.91 mL,62.8 mmol) was then added dropwise. The resulting suspension was left tostir at room temperature for 16 h. Propan-2-ol (5 mL) was carefullyadded to the reaction mixture followed by dichloromethane (50 mL) andwashed with distilled H₂O (50 mL×2) and brine (50 mL×2). Dried overNa₂SO₄ and solvent removed in vacuo to leave a red/orange oil. Columnchromatography (hexane/dichloromethane 50:50) eluted the title compoundas a light yellow oil (5.65 g, 83%); R_(f)0.38 (hexane/dichloromethane50:50), c.f. 0.26 (3-Bromo-5-methyl-phenyl)acetonitrile; ¹H NMR (270MHz, CDCl₃) δ 1.68 (6H, s, ArC(CH₃)₂CN), 2.33 (3H, s, ArCH₃), 7.20 (1H,s, ArH), 7.26 (1H, s, ArH) and 7.34 (1H, s, ArH); ¹³C NMR (100.5 MHz,CDCl₃) δ 21.3 (CH₃), 29.0 (CH₃), 36.9 (C), 122.8, 124.1, 124.9, 125.2,131.6, 140.9 and 143.4; HPLC (80% CH₃CN in H₂O) t_(r)=2.600 (89.65%);LCMS (APCI), m/z 239.93 (⁸¹BrM⁺, 3%), 237.93 (⁷⁹BrM⁺, 4), 212.92(⁸¹BrM⁺−CN, 100), 210.92 (⁷⁹BrM⁺−CN, 96), 157.89 (M⁺−Br, 18).

2-(3-Bromo-5-bromomethyl-phenyl)-2-methylpropionitrile (TJA01036)

C₁₁H₁₁Br₂N MW 317.03

To a solution of sodium bromate (9.51 g, 63.0 mmol) in distilled H₂O (32mL) was added

TJA01035 (5.00 g, 21.0 mmol) in cyclohexane (42 mL). To this clearmixture a solution of sodium hydrogen sulphate (7.56 g, 63.0 mmol) indistilled H₂O (63 mL) was added drop wise with vigorous stirring over 1h. The reaction mixture was stirred for a further 4 h at roomtemperature. The cyclohexane was separated and diethyl ether (100 mL)added. This was then washed with saturated Na2SO_(3(aq)) (50 mL),distilled water (50 ml×2) and brine (50 mL×2). Dried over Na₂SO₄ andsolvent removed in vacuo to leave viscous orange oil. Columnchromatography (hexane/dichloromethane 50:50) eluted starting materialand the title compound as a clear viscous oil (3.64 g, 54%), R_(f) 0.55(hexane/dichloromethane 50:50), c.f. 0.38(2-(3-bromo-5-methylphenyl)-2-methyl-propionitrile); ¹H NMR (270 MHz,CDCl₃) δ 1.71 (6H, s, ArC(CH₃)₂CN), 4.41 (2H, s, ArCH₂Br), 7.40-7.41(1H, t, J=1.7, ArH) and 7.48-7.51 (2H, m, ArH); HPLC (80% CH₃CN in H₂O)t_(r)=2.508 (83.05%); LRMS (FAB⁺), m/z 319.1 (⁸¹BrM⁺, 100%), 317.1(⁷⁹BrM⁺, 100).

2-(3-Bromo-5-[1,2,4-triazole-1-yl-methylphenyl)-2-methylpropionitrile(TJA01038, STX1453)

C₁₃H₁₃BrN₄ MW 305.18

TJA01036 (3.20 g, 10.1 mmol), 1,2,4-triazole (1.05 g, 15.2 mmol),potassium carbonate (1.40 g, 10.1 mmol), potassium iodide (0.10 g, 0.600mmol) and acetone (150 mL) were loaded to an r.b. flask. With vigorousstirring this mixture was set to reflux (60° C.) for 24 h. The reactionmixture was allowed to cool and acetone was removed in vacuo. Theresidues were taken up in ethyl acetate (50 mL) and washed withdistilled water (50 mL×2), 1M NaOH (50 mL×1) and brine (50 mL×2). Driedover Na₂SO₄ and solvent removed in vacuo to leave a yellow oil. Columnchromatography (ethyl acetate) eluted the title compound as a clearviscous oil that crystallised on standing to give a colourlesscrystalline solid (1.97 g, 64%), mp 70.9-71.8° C.; R_(f) 0.24 (ethylacetate).

¹H NMR (270 MHz, CDCl₃) δ 1.68 (6H, s, ArC(CH₃)₂CN), 5.33 (2H, s,ArCH₂N), 7.40-7.41 (2H, t, J=1.7, ArH), 7.54-7.55 (1H, t, J=1.7, ArH),7.99 (1H, s, C₂H₂N₃) and 8.12 (1H, s, C₂H₂N₃); ¹³C NMR (100.5 MHz,CDCl₃) δ 29.0 (CH₃), 37.0 (C), 52.6 (CH₂), 123.5, 123.7, 128.6, 130.4,137.7, 143.4, 144.5 and 152.6 (one overlapping peak); HPLC (60% CH₃CN inH₂O increasing to 95% over 10 min) t_(r)=2.293 (98.87%); MS (EI), m/z307.09 (⁸¹BrM⁺, 100%), 305.09 (⁷⁹BrM⁺, 99), 238.01 ((⁸¹BrM−(C₂H₂N₃)⁺,22), 236.01 ((⁷⁹BrM−(C₂H₂N₃)⁺, 24).

2-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-(4-hydroxystyryl)phenyl)-2-methylpropanenitrile(TJA01180)

C₂₁H₂₀N₄O MW 344.41

TJA01037 (0.100 g, 0.328 mmol), 4-vinylphenol [10% wt in propyleneglycol] (0.787 g, 0.655 mmol), triethylamine (91.0 μL, 0.655 mmol) andtriphenylphosphine (0.029 g, 0.112 mmol) were loaded to a dry 10 mL r.b.flask purged with N_(2 (g)). Anhydrous THF (1.5 mL) was added via asyringe and the resulting clear solution degassed via bubbling N_(2 (g))for 30 min. Pd (OAc)₂ (0.012 g, 0.056 mmol) was added, a condenserattached and the reaction mixture heated to 85° C. for 22 h withconstant stirring (4-vinylphenol (0.394 g, 0.328 mmol) added after 6 h).After the reaction had been allowed to cool THF was removed in vacuo andthe residues dissolved in dichloromethane (20 mL), washed with 2MKHSO_(4 (aq)) (20 mL), distilled H₂O (20 mL×3) and brine (20 mL). Theorganic portion was dried over MgSO₄ and solvent removed in vacuo.Column chromatography (ethyl acetate) eluted a colourless viscous oil(0.080 g, 71%) which was used without further purification.

4-(3-((1H-1,2,4-triazol-1-yl)methyl)-5-(2-cyanopropan-2-yl)styryl)phenylsulfamate (TJA01181, STX1833)

C₂₁H₂₁N₅O₃S MW 423.49

Sulfamoyl chloride in toluene (0.60 M, 1.93 mL) was transferred to a 10mL r.b. flask and the solvent removed under vacuum at 30° C. On coolinga white solid formed to which was added N,N-dimethylacetamide (1.5 mL)to form a colourless solution. TJA01180 (0.080 g, 0.232 mmol) was addedand the solution left to stir at room temperature under N_(2 (g)) for 60h. The reaction mixture was then poured into distilled H₂O (25 mL) andextracted with ethyl acetate (25 mL×2). The organic layers were combinedand washed with distilled H₂O (25 mL×4) and brine (25 mL). Dried overMgSO₄ and solvent removed in vacuo to leave off white residues. Columnchromatography (dichloromethane/acetone 80:20) eluted the title compoundas a white solid (0.035 g, 36%), mp 151.4-152.1° C.; R_(f): 0.30(dichloromethane/acetone 75:25); ¹H NMR (600 MHz, DMSO-d₆) δ 1.73 (6H,s, ArC(CH₃)₂)CN), 5.50 (2H, s, ArCH₂N), 7.30-7.33 (3H, m, ArH & vinyl),7.35-7.38 (1H, d, J=16.6 Hz, trans vinyl), 7.31-7.33, 7.42 (1H, s, ArH),7.51 (1H, s, ArH), 7.71 (1H, s, ArH), 7.72-7.74 (2H, d, J=8.4 Hz,AA′BB′), 8.04 (1H, s, C₂H₂N₃), 8.06 (2H, bs, ArOSO₂NH₂) and 8.73 (1H, s,C₂H₂N₃); HPLC (90% CH₃CN in H₂O) t_(r)=1.784 (99.10%); LCMS (APCI), m/z424.42 (M⁺+H, 100%), 345.47 ((M⁺+H)—OSO₂NH₂, 27).

Biological Data

A number of compounds (including anastrozole, STX1022 and STX1023, thestructures of which are shown below) were tested for aromatase andsteroid sulphatase inhibition in accordance with the above Protocols.

In vivo data were recorded using the above described aromatase and STSanimals assays. The relevant compounds were administered and for eachanimal both aromatase and STS activities were determined. % AROMATASE %STS STX INHIBITION INHIBITION CODE STRUCTURE (10 μM) (10 μM) STX 1522

98.0 ± 0.3% 96.1 ± 0.3% STX 1523

99.7 ± 0.2% 24.4 ± 3.1% STX 1528

83.4 ± 1.3% <10% STX 1729

99.9 ± 0.2% 82.3 ± 0.8% STX 1731

99.5 ± 0.2% 21.4 ± 7.9% STX 1732

99.8 ± 0.1% <10% STX 1761

99.8 ± 0.2% <10% STX 1794

99.7 ± 0.1% 75.3 ± 0.6% STX 1795

100 ± 0.0% 73.6 ± 0.1% STX 1829

99.5 ± 0.2% 17.6 ± 4.0% STX 1830

52.7 ± 6.7% 33.4 ± 5.3% STX 1833

99.6 ± 0.1% 21.5 ± 4.7%

JEG3 cells IC50 In vivo % Inhibition (nM) (10 mg/kg) Compound AromataseSTS Aromatase (after 3 h) STS STX1023 3.5 >10,000 ND 87 Anastrozole 1.5ND ND ND STX1022 1 — — —

The invention will now be further described by the following numberedparagraphs:

1. A compound of Formula I

wherein

X, Y and Z are each independently of each other an optional linkergroup;

R₁ is a ring system;

R₂ is selected from hydrocarbyl groups, oxyhydrocarbyl groups, cyano(—CN), nitro (—NO₂) and halogens;

R₃ and R₄ are independently selected from H and hydrocarbyl

ring A and B are independently optionally further substituted.

2. A compound according to paragraph 1 of Formula II

3. A compound according to paragraph 1 of Formula III

4. A compound according to paragraph 1 of Formula IV

5. A compound according to paragraph 1 of Formula V

6. A compound according to paragraph 1 of Formula VI

7. A compound according to paragraph 1 of Formula VII

8. A compound according to paragraph 1 of Formula VIII or Formula VIIIa

9. A compound according to paragraph 1 of Formula VIII

10. A compound according to any one of the preceding paragraphs whereinat least one of the optional linker groups is present.

11. A compound according to any one of the preceding paragraphs whereinat least two of the optional linker groups are present.

12 A compound according to any one of the preceding paragraphs whereineach of X, Y and Z are present.

13. A compound according to any one of the preceding paragraphs whereinX is selected from hydrocarbyl, oxyhydrocarbyl, COO, CO, S, O, SO, SO₂,NR, and SO₂NR, wherein R is selected from H and hydrocarbyl groups.

14. A compound according to paragraph 13 wherein the hydrocarbyl groupis a straight of branched alkyl group.

15. A compound according to paragraph 13 wherein the hydrocarbyl groupis a straight chain alkyl group.

16. A compound according to paragraph 13 wherein the oxyhydrocarbylgroup is —O-alkyl-, wherein alkyl is a straight of branched alkyl group.

17. A compound according to paragraph 13 wherein the oxyhydrocarbylgroup is —O-alkyl-, wherein alkyl is a straight chain alkyl group

18. A compound according to any one of the preceding paragraphs whereinX is selected from (CH₂)n, CH═CH (preferably trans configuration),O(CH₂)n, (CH₂)nO, S(CH₂)n, (CH₂)nS, CO(CH₂)n, (CH₂)nCO, CONH(CH₂)n,(CH₂)nCONH, COO(CH₂)n, (CH₂)nCOO, SO(CH₂)n, (CH₂)nSO, SO₂(CH₂)n,(CH₂)nSO₂, SO₂NMe(CH₂)n, (CH₂)nSO₂NMe; SO₂NH(CH₂)n, and (CH₂)nSO₂NH;wherein n is independently an integer from 0 to 6.

19. A compound according to paragraph 18 wherein n is independently aninteger from 1 to 6.

20. A compound according to any one of the preceding paragraphs whereinX is selected from SO₂NH, SO₂NMe, CONH, OCH₂, SCH₂, and CH═CH.

21. A compound according to any one of the preceding paragraphs whereinY is selected from hydrocarbyl, oxyhydrocarbyl, COO, CO, S, O, SO, SO₂,NR, and SO₂NR, wherein R is selected from H and hydrocarbyl groups.

22. A compound according to any one of the preceding paragraphs whereinY is selected from hydrocarbyl, CO, and SO₂.

23. A compound according to paragraph 21 or 22 wherein the hydrocarbylgroup is a straight of branched alkyl group.

24. A compound according to paragraph 21 or 22 wherein the hydrocarbylgroup is a straight chain alkyl group.

25. A compound according to any one of the preceding paragraphs whereinY is selected is from groups selected from (CH₂)m, CO(CH₂)m, (CH₂)mCO,SO₂(CH₂)m and wherein m is independently an integer from 0 to 6.

26. A compound according to paragraph 25 wherein m is independently aninteger from 1 to 6.

27. A compound according to any one of the preceding paragraphs whereinY is (CH₂)m, wherein m is an integer from 1 to 6.

28. A compound according to paragraph 27 wherein Y is —CH₂—.

29. A compound according to any one of the preceding paragraphs whereinZ is selected from hydrocarbyl, oxyhydrocarbyl, COO, CO, S, O, SO, SO₂,NR, and SO₂NR, wherein R is selected from H and hydrocarbyl groups.

30. A compound according to any one of the preceding paragraphs whereinZ is a hydrocarbyl group.

31. A compound according to paragraph 30 wherein the hydrocarbyl groupis a straight of branched alkyl group.

32. A compound according to paragraph 30 wherein the hydrocarbyl groupis a branched alkyl group.

33. A compound according to any one of the preceding paragraphs whereinZ is CpH₂p, wherein p is an integer from 1 to 6.

34. A compound according to paragraph 33 wherein p is independently aninteger from 1 to 3.

35. A compound according to any one of the preceding paragraphs whereinZ is selected from —C(CH₃)₂—, —C(O)O—,

36. A compound according to any one of the preceding paragraphs whereinR₁ is or comprises an aromatic ring.

37. A compound according to any one of the preceding paragraphs whereinR₁ is selected from substituted and unsubstituted aromatic rings.

38. A compound according to any one of the preceding paragraphs whereinR₁ is selected from ring systems comprising from 3 to 10 members.

39. A compound according to any one of the preceding paragraphs whereinR₁ is selected from ring systems comprising from 5, 6 or 7 members. 40.A compound according to any one of the preceding paragraphs wherein R₁is selected from ring systems comprising carbon and optionally one ormore hetero atoms.

41. A compound according to any one of the preceding paragraphs whereinR₁ is selected from ring systems comprising carbon and optionally one,two or three hetero atoms.

42. A compound according to any one of the preceding paragraphs whereinR₁ is selected from ring systems comprising carbon and one or morehetero atoms.

43. A compound according to any one of the preceding paragraphs whereinR₁ is selected from ring systems comprising carbon and one or morehetero atoms selected from Nitrogen, Sulphur and Oxygen.

44. A compound according to any one of the preceding paragraphs whereinR₁ is selected from heterocyclic ring systems, wherein the ringcomprises carbon and nitrogen.

45. A compound according to any one of the preceding paragraphs whereinR₁ is selected from 4H-1,2,4-triazole, 1H-1,2,4-triazole and1H-1,2,3-triazole.

46. A compound according to any one of the preceding paragraphs whereinR₁ is 1H-1,2,4-triazole.

47. A compound according to any one of the preceding paragraphs whereinR₁ is

48. A compound according to any one of the preceding paragraphs whereinR₂ is selected from hydrocarbyl groups, oxyhydrocarbyl groups, cyano(—CN), nitro (—NO₂) and halogens, wherein the hydrocarbyl group is astraight of branched alkyl group.

49. A compound according to paragraph 48 wherein the hydrocarbyl groupis a straight chain alkyl group.

50. A compound according to paragraph 48 wherein the hydrocarbyl groupis (CH₂)qCH₃, wherein q is an integer from 0 to 6.

51. A compound according to any one of the preceding paragraphs whereinR₂ is selected from hydrocarbyl groups, oxyhydrocarbyl groups, cyano(—CN), nitro (—NO₂) and halogens, wherein the oxyhydrocarbyl group is—O-alkyl-, wherein alkyl is a straight or branched alkyl group.

52. A compound according to paragraph 51 wherein the oxyhydrocarbylgroup is —O-alkyl-, wherein alkyl is a straight chain alkyl group

53. A compound according to paragraph 52 wherein the oxyhydrocarbylgroup is —O(CH₂)rCH₃, wherein r is an integer from 0 to 6.

54. A compound according to any one of the preceding paragraphs whereinR₂ is selected from —CH₃, —OCH₃, cyano (—CN), nitro (—NO₂) and halogens.

55. A compound according to any one of the preceding paragraphs whereinR₃ and R₄ are independently selected from H, alkyl, cycloalkyl, alkenyl,acyl and aryl, or combinations thereof, or together represent alkylene,wherein the or each alkyl or cycloalkyl or alkenyl or optionally containone or more hetero atoms or groups.

56. A compound according to any one of the preceding paragraphs whereinat least one of R₃ and R₄ is H.

57. A compound according to any one of the preceding paragraphs whereinR₃ is H and R₄ is H.

58. A compound according to any one of the preceding paragraphs whereinrings A and B are independently not further substituted.

59. A compound according to any one of the preceding paragraphs whereinneither ring A nor ring B is further substituted.

60. A compound according to any one paragraphs 1 to 57 wherein rings Aand B are independently further substituted by groups selected fromhydrocarbyl groups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) andhalogens.

61. A compound according to any one paragraphs 1 to 57 wherein rings Aand B are independently further substituted by groups selected from C1-6alkyl groups, C1-6 alkoxy groups, cyano (—CN), nitro (—NO₂) andhalogens.

62. A compound according to any one paragraphs 1 to 57 wherein rings Aand B are independently further substituted by groups selected from—CH₃, —CH₂CH₃, —OCH₃, cyano (—CN), nitro (—NO₂) and halogens.

63. A compound according to paragraph 1 selected from compounds of theformulae

64. A compound according to any one of paragraphs 1 to 63 for use inmedicine.

65. A pharmaceutical composition comprising the compound according toany one of paragraphs 1 to 63 optionally admixed with a pharmaceuticallyacceptable carrier, diluent, excipient or adjuvant.

66. Use of a compound according to any one of paragraphs 1 to 63 in themanufacture of a medicament for use in the therapy of a condition ordisease associated with STS and/or aromatase and/or cell cycling and/orapoptosis and/or cell growth.

67. Use of a compound according to any one of paragraphs 1 to 63 in themanufacture of a medicament for use in the therapy of a condition ordisease associated with STS and aromatase.

68. Use of a compound according to any one of paragraphs 1 to 63 in themanufacture of a medicament for use in the therapy of a condition ordisease associated with adverse STS levels and/or adverse aromataselevels and/or cell cycling and/or apoptosis and/or cell growth.

69. Use of a compound according to any one of paragraphs 1 to 63 in themanufacture of a medicament for use in the therapy of a condition ordisease associated with adverse STS levels and adverse aromatase levels.

70. Use of a compound according to any one of paragraphs 1 to 63 in themanufacture of a medicament for inhibiting STS activity and/orinhibiting aromatase activity.

71. Use of a compound according to any one of paragraphs 1 to 63 in themanufacture of a medicament for inhibiting STS activity and inhibitingaromatase activity.

72. A compound as substantially hereinbefore described with reference tothe Examples.

73. A composition as substantially hereinbefore described with referenceto the Examples.

74. A method or use as substantially hereinbefore described withreference to the Examples.

All publications and patents and patent applications mentioned in theabove specification are herein incorporated by reference. Variousmodifications and variations of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin chemistry, biology or related fields are intended to be within thescope of the following claims.

1. A compound of Formula I

wherein X, Y and Z are each independently of each other an optionallinker group; R₁ is a ring system; R₂ is selected from hydrocarbylgroups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) and halogens;R₃ and R₄ are independently selected from H and hydrocarbyl ring A and Bare independently optionally further substituted.
 2. A compoundaccording to claim 1 of Formula II


3. A compound according to claim 1 wherein at least one or at least twoof the optional linker groups is present.
 4. A compound according toclaim 1 wherein each of X, Y and Z are present.
 5. A compound accordingto claim 1 wherein X is selected from hydrocarbyl, oxyhydrocarbyl, COO,CO, S, O, SO, SO₂, NR, and SO₂NR, wherein R is selected from H andhydrocarbyl groups.
 6. A compound according to claim 5 wherein thehydrocarbyl group is a straight or branched alkyl group.
 7. A compoundaccording to claim 5 wherein the hydrocarbyl group is a straight chainalkyl group.
 8. A compound according to claim 5 wherein theoxyhydrocarbyl group is —O-alkyl-, wherein alkyl is a straight orbranched alkyl group.
 9. A compound according to claim 5 wherein theoxyhydrocarbyl group is —O-alkyl-, wherein alkyl is a straight chainalkyl group
 10. A compound according to claim 1 wherein X is selectedfrom (CH₂)n, CH═CH (preferably trans configuration), O(CH₂)n, (CH₂)nO,S(CH₂)n, (CH₂)nS, CO(CH₂)n, (CH₂)nCO, CONH(CH₂)n, (CH₂)nCONH, COO(CH₂)n,(CH₂)nCOO, SO(CH₂)n, (CH₂)nSO, SO₂(CH₂)n, (CH₂)nSO₂, SO₂NMe(CH₂)n,(CH₂)nSO₂NMe; SO₂NH(CH₂)n, and (CH₂)nSO₂NH; wherein n is independentlyan integer from 0 to
 6. 11. A compound according to claim 10 wherein nis independently an integer from 1 to
 6. 12. A compound according toclaim 1 wherein X is selected from SO₂NH, SO₂NMe, CONH, OCH₂, SCH₂, andCH═CH.
 13. A compound according to claim 1 wherein Y is selected fromhydrocarbyl, oxyhydrocarbyl, COO, CO, S, O, SO, SO₂, NR, and SO₂NR,wherein R is selected from H and hydrocarbyl groups.
 14. A compoundaccording to claim 1 wherein Y is selected from hydrocarbyl, CO, andSO₂.
 15. A compound according to claim 13 or 14 wherein the hydrocarbylgroup is a straight or branched alkyl group.
 16. A compound according toclaim 13 or 14 wherein the hydrocarbyl group is a straight chain alkylgroup.
 17. A compound according to claim 1 wherein Y is selected fromgroups selected from (CH₂)m, CO(CH₂)m, (CH₂)mCO, SO₂(CH₂)m and wherein mis independently an integer from 0 to
 6. 18. A compound according toclaim 17 wherein m is independently an integer from 1 to
 6. 19. Acompound according to claim 1 wherein Y is (CH₂)m, wherein m is aninteger from 1 to
 6. 20. A compound according to claim 19 wherein Y is—CH₂—.
 21. A compound according to claim 1 wherein Z is selected fromhydrocarbyl, oxyhydrocarbyl, COO, CO, S, O, SO, SO₂, NR, and SO₂NR,wherein R is selected from H and hydrocarbyl groups.
 22. A compoundaccording to claim 1 wherein Z is a hydrocarbyl group.
 23. A compoundaccording to claim 22 wherein the hydrocarbyl group is a straight ofbranched alkyl group.
 24. A compound according to claim 22 wherein thehydrocarbyl group is a branched alkyl group.
 25. A compound according toclaim 1 wherein Z is CpH₂p, wherein p is an integer from 1 to
 6. 26. Acompound according to claim 25 wherein p is independently an integerfrom 1 to
 3. 27. A compound according to claim 1 wherein Z is selectedfrom —C(CH₃)₂—, —C(O)O—,


28. A compound according to claim 1 wherein R₁ is or comprises anaromatic ring.
 29. A compound according to claim 1 wherein R₁ isselected from substituted and unsubstituted aromatic rings.
 30. Acompound according to claim 1 wherein R₁ is selected from ring systemscomprising from 3 to 10 members.
 31. A compound according to claim 1wherein R₁ is selected from ring systems comprising from 5, 6 or 7members.
 32. A compound according to claim 1 wherein R₁ is selected fromring systems comprising carbon and optionally one or more hetero atoms.33. A compound according to claim 1 wherein R₁ is selected from ringsystems comprising carbon and optionally one, two or three hetero atoms.34. A compound according to claim 1 wherein R₁ is selected from ringsystems comprising carbon and one or more hetero atoms.
 35. A compoundaccording to claim 1 wherein R₁ is selected from ring systems comprisingcarbon and one or more hetero atoms selected from Nitrogen, Sulphur andOxygen.
 36. A compound according to claim 1 wherein R₁ is selected fromheterocyclic ring systems, wherein the ring comprises carbon andnitrogen.
 37. A compound according to claim 1 wherein R₁ is selectedfrom 4H-1,2,4-triazole, 1H-1,2,4-triazole and 1H-1,2,3-triazole.
 38. Acompound according to claim 1 wherein R₁ is 1H-1,2,4-triazole.
 39. Acompound according to claim 1 wherein R₁ is


40. A compound according to claim 1 wherein R₂ is selected fromhydrocarbyl groups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) andhalogens, wherein the hydrocarbyl group is a straight of branched alkylgroup.
 41. A compound according to claim 40 wherein the hydrocarbylgroup is a straight chain alkyl group.
 42. A compound according to claim40 wherein the hydrocarbyl group is (CH₂)qCH₃, wherein q is an integerfrom 0 to
 6. 43. A compound according to claim 1 wherein R₂ is selectedfrom hydrocarbyl groups, oxyhydrocarbyl groups, cyano (—CN), nitro(—NO₂) and halogens, wherein the oxyhydrocarbyl group is —O-alkyl-,wherein alkyl is a straight or branched alkyl group.
 44. A compoundaccording to claim 43 wherein the oxyhydrocarbyl group is —O-alkyl-,wherein alkyl is a straight chain alkyl group
 45. A compound accordingto claim 44 wherein the oxyhydrocarbyl group is —O(CH₂)rCH₃, wherein ris an integer from 0 to
 6. 46. A compound according to claim 1 whereinR₂ is selected from —CH₃, —OCH₃, cyano (—CN), nitro (—NO₂) and halogens.47. A compound according to claim 1 wherein R₃ and R₄ are independentlyselected from H, alkyl, cycloalkyl, alkenyl, acyl and aryl, orcombinations thereof, or together represent alkylene, wherein the oreach alkyl or cycloalkyl or alkenyl or optionally contain one or morehetero atoms or groups.
 48. A compound according to claim 1 wherein atleast one of R₃ and R₄ is H.
 49. A compound according to claim 1 whereinR₃ is H and R₄ is H.
 50. A compound according to claim 1 wherein rings Aand B are independently not further substituted.
 51. A compoundaccording to claim 1 wherein neither ring A nor ring B is furthersubstituted.
 52. A compound according to claim 1 wherein rings A and Bare independently further substituted by groups selected fromhydrocarbyl groups, oxyhydrocarbyl groups, cyano (—CN), nitro (—NO₂) andhalogens.
 53. A compound according to claim 1 wherein rings A and B areindependently further substituted by groups selected from C1-6 alkylgroups, C1-6 alkoxy groups, cyano (—CN), nitro (—NO₂) and halogens. 54.A compound according to claim 1 wherein rings A and B are independentlyfurther substituted by groups selected from —CH₃, —CH₂CH₃, —OCH₃, cyano(—CN), nitro (—NO₂) and halogens.
 55. A compound according to claim 1selected from compounds of the formulae


56. A pharmaceutical composition comprising the compound according toclaim 1 optionally admixed with a pharmaceutically acceptable carrier,diluent, excipient or adjuvant.
 57. A method of treating or preventing acondition or disease associated with STS and/or aromatase and/or cellcycling and/or apoptosis and/or cell growth comprising administering, toa patient in need thereof, an effective amount of a compound accordingto claim 1
 58. A method of treating or preventing a condition or diseaseassociated with STS and aromatase comprising administering, to a patientin need thereof, an effective amount of a compound according to claim 1.59. A method of treating or preventing a condition or disease associatedwith adverse STS levels and/or adverse aromatase levels and/or cellcycling and/or apoptosis and/or cell growth comprising administering, toa patient in need thereof, an effective amount of a compound accordingto claim
 1. 60. A method of treating or preventing a condition ordisease associated with adverse STS levels and adverse aromatase levelscomprising administering, to a patient in need thereof, an effectiveamount of a compound according to claim
 1. 61. A method of inhibitingSTS activity and/or inhibiting aromatase activity comprisingadministering, to a patient in need thereof, an effective amount of acompound according to claim
 1. 62. A method of inhibiting STS activityand inhibiting aromatase activity comprising administering, to a patientin need thereof, an effective amount of a compound according to claim 1.